26 results on '"Linda De Vooght"'
Search Results
2. Characterization of novel double-reporter strains of Mycobacterium abscessus for drug discovery: a study in mScarlet
- Author
-
Clara M. Bento, Kevin van Calster, Tatiana Piller, Gabriel S. Oliveira, Linda de Vooght, Davie Cappoen, Paul Cos, M. Salomé Gomes, and Tânia Silva
- Subjects
Mycobacterium ,drug screening ,fluorescence ,macrophages ,infection models ,reporter strains ,Microbiology ,QR1-502 - Abstract
ABSTRACT Mycobacterium abscessus (Mab) is an emerging pathogen that poses a severe health threat, especially in people with cystic fibrosis and other chronic lung diseases. Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used in vitro for identifying hits in a high-throughput manner and in vivo to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to in vivo infection (using the Galleria mellonella model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way.IMPORTANCEMycobacterium abscessus (Mab) is currently considered an “incurable nightmare.” Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between in vitro susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as in vitro drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success.
- Published
- 2024
- Full Text
- View/download PDF
3. Development and validation of a multiplex electrochemiluminescence immunoassay to evaluate dry eye disease in rat tear fluids
- Author
-
Agnese Compagnone, An Matheeussen, Linda De Vooght, and Paul Cos
- Subjects
Medicine ,Science - Abstract
Abstract Dry eye disease (DED) is a challenge in ophthalmology. Rat models represent valuable tools to study the pathophysiology and to develop novel treatments. A major challenge in DED research is detecting multiple biomarkers in a low tear volume sample. Multiplex immunoassays for DED rat research are missing. We have developed a multiplex electrochemiluminescence immunoassay (ECLIA) to detect three biomarkers for DED: MMP-9, IL-17 and ICAM-1. Tears, used as matrix, were collected from six healthy Wistar rats. Assays were run based on the U-Plex Meso Scale Diagnostics (MSD) platform, by two independent operators according to the EMA guideline on bioanalytical method validation. Linear mixed, regression models were fit to perform the statistical analysis on the range of concentrations for the chosen analytes. During optimization, it has observed that incubation time, temperature and agitation affected the robustness of the protocol. ECLIA optimum conditions include the use of antibodies at 0.5 µg/ml concentration and 1 h incubation at room temperature with shaking. Precision met the acceptance criteria in the chosen range: 1062–133 pg/ml for ICAM-1, 275–34.4 pg/ml for IL-17, 1750–219 pg/ml for MMP-9. Accuracy and linearity were acceptable for a broader range. This is the first report of a validated ECLIA that allows measurements of three relevant DED biomarkers in rat tear fluids.
- Published
- 2023
- Full Text
- View/download PDF
4. A high-throughput target-based screening approach for the identification and assessment of Mycobacterium tuberculosis mycothione reductase inhibitors
- Author
-
Natalia Smiejkowska, Lauren Oorts, Kevin Van Calster, Linda De Vooght, Rob Geens, Henri-Philippe Mattelaer, Koen Augustyns, Sergei V. Strelkov, Dirk Lamprecht, Koen Temmerman, Yann G.-J. Sterckx, Davie Cappoen, and Paul Cos
- Subjects
high-throughput screening ,mycothione reductase ,tuberculosis ,drug discovery ,Microbiology ,QR1-502 - Abstract
ABSTRACTThe World Health Organization’s goal to combat tuberculosis (TB) is hindered by the emergence of anti-microbial resistance, therefore necessitating the exploration of new drug targets. Multidrug regimens are indispensable in TB therapy as they provide synergetic bactericidal effects, shorten treatment duration, and reduce the risk of resistance development. The research within our European RespiriTB consortium explores Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we describe the development and validation of a luminescence-coupled, target-based assay for the identification of novel compounds inhibiting Mycobacterium tuberculosis mycothione reductase (MtrMtb), an enzyme with a role in the protection against oxidative stress. Recombinant MtrMtb was employed for the development of a highly sensitive, robust high-throughput screening (HTS) assay by coupling enzyme activity to a bioluminescent readout. Its application in a semi-automated setting resulted in the screening of a diverse library of ~130,000 compounds, from which 19 hits were retained after an assessment of their potency, selectivity, and specificity. The selected hits formed two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens.IMPORTANCEThe growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.
- Published
- 2024
- Full Text
- View/download PDF
5. Selected strategies to fight pathogenic bacteria
- Author
-
Aiva Plotniece, Arkadij Sobolev, Claudiu T. Supuran, Fabrizio Carta, Fredrik Björkling, Henrik Franzyk, Jari Yli-Kauhaluoma, Koen Augustyns, Paul Cos, Linda De Vooght, Matthias Govaerts, Juliana Aizawa, Päivi Tammela, and Raivis Žalubovskis
- Subjects
Antimicrobials ,natural products ,metalloenzymes ,biopharmaceuticals ,biofilms ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems.
- Published
- 2023
- Full Text
- View/download PDF
6. In-depth biological characterization of two black soldier fly anti-Pseudomonas peptides reveals LPS-binding and immunomodulating effects
- Author
-
Laurence Van Moll, Milan Wouters, Jeroen De Smet, Linda De Vooght, Peter Delputte, Mik Van Der Borght, and Paul Cos
- Subjects
Pseudomonas aeruginosa ,antimicrobial peptides ,LPS binding ,black soldier fly ,lipopolysaccharide ,Microbiology ,QR1-502 - Abstract
ABSTRACT As effector molecules of the innate immune system, antimicrobial peptides (AMPs) have gathered substantial interest as a potential future generation of antibiotics. Here, we demonstrate the anti-Pseudomonas activity and lipopolysaccharide (LPS)-binding ability of HC1 and HC10, two cecropin peptides from the black soldier fly (Hermetia Illucens). Both peptides are active against a wide range of Pseudomonas aeruginosa strains, including drug-resistant clinical isolates. Moreover, HC1 and HC10 can bind to lipid A, the toxic center of LPS and reduce the LPS-induced nitric oxide and cytokine production in murine macrophage cells. This suggests that the peptide-LPS binding can also lower the strong inflammatory response associated with P. aeruginosa infections. As the activity of AMPs is often influenced by the presence of salts, we studied the LPS-binding activity of HC1 and HC10 in physiological salt concentrations, revealing a strong decrease in activity. Our research confirmed the early potential of HC1 and HC10 as starting points for anti-Pseudomonas drugs, as well as the need for structural or formulation optimization before further preclinical development can be considered. IMPORTANCE The high mortality and morbidity associated with Pseudomonas aeruginosa infections remain an ongoing challenge in clinical practice that requires urgent action. P. aeruginosa mostly infects immunocompromised individuals, and its prevalence is especially high in urgent care hospital settings. Lipopolysaccharides (LPSs) are outer membrane structures that are responsible for inducing the innate immune cascade upon infection. P. aeruginosa LPS can cause local excessive inflammation, or spread systemically throughout the body, leading to multi-organ failure and septic shock. As antimicrobial resistance rates in P. aeruginosa infections are rising, the research and development of new antimicrobial agents remain indispensable. Especially, antimicrobials that can both kill the bacteria themselves and neutralize their toxins are of great interest in P. aeruginosa research to develop as the next generation of drugs.
- Published
- 2023
- Full Text
- View/download PDF
7. In Vitro Persistence Level Reflects In Vivo Antibiotic Survival of Natural Pseudomonas aeruginosa Isolates in a Murine Lung Infection Model
- Author
-
Laure Verstraete, Juliana Aizawa, Matthias Govaerts, Linda De Vooght, Rob Lavigne, Jan Michiels, Bram Van den Bergh, and Paul Cos
- Subjects
persistence ,antibiotics ,tolerance ,Pseudomonas aeruginosa ,in vivo model ,Microbiology ,QR1-502 - Abstract
ABSTRACT Clinicians are increasingly confronted with the limitations of antibiotics to clear bacterial infections in patients. It has long been assumed that only antibiotic resistance plays a pivotal role in this phenomenon. Indeed, the worldwide emergence of antibiotic resistance is considered one of the major health threats of the 21st century. However, the presence of persister cells also has a significant influence on treatment outcomes. These antibiotic-tolerant cells are present in every bacterial population and are the result of the phenotypic switching of normal, antibiotic-sensitive cells. Persister cells complicate current antibiotic therapies and contribute to the development of resistance. In the past, extensive research has been performed to investigate persistence in laboratory settings; however, antibiotic tolerance under conditions that mimic the clinical setting remain poorly understood. In this study, we optimized a mouse model for lung infections with the opportunistic pathogen Pseudomonas aeruginosa. In this model, mice are intratracheally infected with P. aeruginosa embedded in seaweed alginate beads and subsequently treated with tobramycin via nasal droplets. A diverse panel of 18 P. aeruginosa strains originating from environmental, human, and animal clinical sources was selected to assess survival in the animal model. Survival levels were positively correlated with the survival levels determined via time-kill assays, a common method to study persistence in the laboratory. We showed that survival levels are comparable and thus that the classical persister assays are indicative of antibiotic tolerance in a clinical setting. The optimized animal model also enables us to test potential antipersister therapies and study persistence in relevant settings. IMPORTANCE The importance of targeting persister cells in antibiotic therapies is becoming more evident, as these antibiotic-tolerant cells underlie relapsing infections and resistance development. Here, we studied persistence in a clinically relevant pathogen, Pseudomonas aeruginosa. It is one of the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.), which are considered major health threats. P. aeruginosa is mostly known to cause chronic lung infections in cystic fibrosis patients. We mimicked these lung infections in a mouse model to study persistence under more clinical conditions. It was shown that the survival levels of natural P. aeruginosa isolates in this model are positively correlated with the survival levels measured in classical persistence assays in vitro. These results not only validate the use of our current techniques to study persistence but also open opportunities to study new persistence mechanisms or evaluate new antipersister strategies in vivo.
- Published
- 2023
- Full Text
- View/download PDF
8. Antibiotic Tolerance Indicative of Persistence Is Pervasive among Clinical Streptococcus pneumoniae Isolates and Shows Strong Condition Dependence
- Author
-
Nele Geerts, Linda De Vooght, Ioannis Passaris, Peter Delputte, Bram Van den Bergh, and Paul Cos
- Subjects
Streptococcus pneumoniae ,autolysis ,persistence ,antibiotics ,tolerance ,Microbiology ,QR1-502 - Abstract
ABSTRACT Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem, as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. In this study, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which assumes that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof of concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections, which will set the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure, and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.
- Published
- 2022
- Full Text
- View/download PDF
9. Targeting the tsetse-trypanosome interplay using genetically engineered Sodalis glossinidius.
- Author
-
Linda De Vooght, Karin De Ridder, Shahid Hussain, Benoît Stijlemans, Patrick De Baetselier, Guy Caljon, and Jan Van Den Abbeele
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Sodalis glossinidius, a secondary bacterial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components interfering with African trypanosome transmission (i.e. paratransgenesis). Nanobodies (Nbs) have been proposed as potential candidates to target the parasite during development in the tsetse fly. In this study, we have generated an immune Nb-library and developed a panning strategy to select Nbs against the Trypanosoma brucei brucei procyclic developmental stage present in the tsetse fly midgut. Selected Nbs were expressed, purified, assessed for binding and tested for their impact on the survival and growth of in vitro cultured procyclic T. b. brucei parasites. Next, we engineered S. glossinidius to express the selected Nbs and validated their ability to block T. brucei development in the tsetse fly midgut. Genetically engineered S. glossinidius expressing Nb_88 significantly compromised parasite development in the tsetse fly midgut both at the level of infection rate and parasite load. Interestingly, expression of Nb_19 by S. glossinidius resulted in a significantly enhanced midgut establishment. These data are the first to show in situ delivery by S. glossinidius of effector molecules that can target the trypanosome-tsetse fly crosstalk, interfering with parasite development in the fly. These proof-of-principle data represent a major step forward in the development of a control strategy based on paratransgenic tsetse flies. Finally, S. glossinidius-based Nb delivery can also be applied as a powerful laboratory tool to unravel the molecular determinants of the parasite-vector association.
- Published
- 2022
- Full Text
- View/download PDF
10. Inhibitory Effect on Nitric Oxide Release in LPS-Stimulated Macrophages and Free Radical Scavenging Activity of Croton linearis Jacq. Leaves
- Author
-
Jesús García Díaz, Rosalia González Fernández, Julio César Escalona Arranz, Gabriel Llauradó Maury, Daniel Méndez Rodríguez, Linda De Vooght, Enrique Molina, Emmy Tuenter, Luc Pieters, and Paul Cos
- Subjects
Croton linearis ,nitric oxide release ,alkaloids ,flavonoids ,antioxidants ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Oxidative stress is an important component of many diseases including cancer, along with inflammatory and neurodegenerative processes. Natural antioxidants have emerged as promising substances to protect the human body against reactive oxygen and nitrogen species. The present study evaluates the inhibition of nitric oxide (NO) production in LPS-stimulated RAW 264.7 murine macrophages and the free radical scavenging activity of Croton linearis Jacq. leaves. UPLC-QTOF-MS analysis identified 18 compounds: nine alkaloids with a morphinane, benzylisoquinoline or aporphine nucleus, and nine O-glycosylated-flavonoids with quercetin, kaempferol and isorhamnetin as the aglycones. The crude extract (IC50 21.59 µg/mL) and the n-hexane fraction (IC50 4.88 µg/mL) significantly reduced the NO production in LPS-stimulated macrophages but with relatively high cytotoxicity (CC50 75.30 and CC50 70.12 µg/mL, respectively), while the ethyl acetate fraction also showed good activity (IC50 40.03 µg/mL) without affecting the RAW 264.7 cell viability. On the other hand, the crude extract, as well as the dichloromethane and ethyl acetate fractions, showed better DPPH and ABTS free radical scavenging activities. Considering the chemical composition and the activity observed for Croton linearis leaves, they may be considered a good source of antioxidants to combat oxidative damage-related diseases.
- Published
- 2022
- Full Text
- View/download PDF
11. Enhancing vector refractoriness to trypanosome infection: achievements, challenges and perspectives
- Author
-
Henry M Kariithi, Irene K Meki, Daniela I Schneider, Linda De Vooght, Fathiya M Khamis, Anne Geiger, Guler Demirbaş-Uzel, Just M Vlak, ikbal Agah iNCE, Sorge Kelm, Flobert Njiokou, Florence N Wamwiri, Imna I Malele, Brian L Weiss, and Adly M M Abd-Alla
- Subjects
Glossina ,Microbiota ,Paratransgenesis ,Vector competence ,Trypanosoma-refractoriness, sterile insect technique ,Hytrosaviridae ,Microbiology ,QR1-502 - Abstract
Abstract With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013–2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP’s major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.
- Published
- 2018
- Full Text
- View/download PDF
12. Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis
- Author
-
Linda De Vooght, Severien Van Keer, and Jan Van Den Abbeele
- Subjects
Paratransgenesis ,Glossina ,Sodalis glossinidius ,Colonization ,Transmission ,GFP ,Microbiology ,QR1-502 - Abstract
Abstract Background Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness. Results In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission. Conclusions Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.
- Published
- 2018
- Full Text
- View/download PDF
13. Combining paratransgenesis with SIT: impact of ionizing radiation on the DNA copy number of Sodalis glossinidius in tsetse flies
- Author
-
Güler Demirbas-Uzel, Linda De Vooght, Andrew G. Parker, Marc J. B. Vreysen, Robert L. Mach, Jan Van Den Abbeele, and Adly M. M. Abd-Alla
- Subjects
Symbiont ,Glossinidae ,Trypanosomosis ,Trypanosomiasis ,Glossina morsitans morsitans ,Microbiology ,QR1-502 - Abstract
Abstract Background Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the causative agents of African Trypanosomosis, which has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. The sterile insect technique (SIT) has shown to be a powerful method to manage tsetse fly populations when used in the frame of an area-wide integrated pest management (AW-IPM) program. To date, the release of sterile males to manage tsetse fly populations has only been implemented in areas to reduce transmission of animal African Trypanosomosis (AAT). The implementation of the SIT in areas with Human African Trypanosomosis (HAT) would require additional measures to eliminate the potential risk associated with the release of sterile males that require blood meals to survive and hence, might contribute to disease transmission. Paratransgenesis offers the potential to develop tsetse flies that are refractory to trypanosome infection by modifying their associated bacteria (Sodalis glossinidius) here after referred to as Sodalis. Here we assessed the feasibility of combining the paratransgenesis approach with SIT by analyzing the impact of ionizing radiation on the copy number of Sodalis and the vectorial capacity of sterilized tsetse males. Results Adult Glossina morsitans morsitans that emerged from puparia irradiated on day 22 post larviposition did not show a significant decline in Sodalis copy number as compared with non-irradiated flies. Conversely, the Sodalis copy number was significantly reduced in adults that emerged from puparia irradiated on day 29 post larviposition and in adults irradiated on day 7 post emergence. Moreover, irradiating 22-day old puparia reduced the copy number of Wolbachia and Wigglesworthia in emerged adults as compared with non-irradiated controls, but the radiation treatment had no significant impact on the vectorial competence of the flies. Conclusion Although the radiation treatment significantly reduced the copy number of some tsetse fly symbionts, the copy number of Sodalis recovered with time in flies irradiated as 22-day old puparia. This recovery offers the opportunity to combine a paratransgenesis approach – using modified Sodalis to produce males refractory to trypanosome infection – with the release of sterile males to minimize the risk of disease transmission, especially in HAT endemic areas. Moreover, irradiation did not increase the vector competence of the flies for trypanosomes.
- Published
- 2018
- Full Text
- View/download PDF
14. The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
- Author
-
Katrien Trappeniers, Irina Matetovici, Jan Van Den Abbeele, and Linda De Vooght
- Subjects
Glossina ,Sodalis glossinidius ,host-symbiont crosstalk ,immune interaction ,transcriptomics ,Microbiology ,QR1-502 - Abstract
Sodalis glossinidius, a vertically transmitted facultative symbiont of the tsetse fly, is a bacterium in the early/intermediate state of its transition toward symbiosis, representing an important model for investigating how the insect host immune defense response is regulated to allow endosymbionts to establish a chronic infection within their hosts without being eliminated. In this study, we report on the establishment of a tsetse fly line devoid of S. glossinidius only, allowing us to experimentally investigate (i) the complex immunological interactions between a single bacterial species and its host, (ii) how the symbiont population is kept under control, and (iii) the impact of the symbiont on the vector competence of the tsetse fly to transmit the sleeping sickness parasite. Comparative transcriptome analysis showed no difference in the expression of genes involved in innate immune processes between symbiont-harboring (GmmSod+) and S. glossinidius-free (GmmSod–) flies. Re-exposure of (GmmSod–) flies to the endosymbiotic bacterium resulted in a moderate immune response, whereas exposure to pathogenic E. coli or to a close non-insect associated relative of S. glossinidius, i.e., S. praecaptivus, resulted in full immune activation. We also showed that S. glossinidius densities are not affected by experimental activation or suppression of the host immune system, indicating that S. glossinidius is resistant to mounted immune attacks and that the host immune system does not play a major role in controlling S. glossinidius proliferation. Finally, we demonstrate that the absence or presence of S. glossinidius in the tsetse fly does not alter its capacity to mount an immune response to pathogens nor does it affect the fly’s susceptibility toward trypanosome infection.
- Published
- 2019
- Full Text
- View/download PDF
15. Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association.
- Author
-
Mauricio Henriques Pontes, Kari Lyn Smith, Linda De Vooght, Jan Van Den Abbeele, and Colin Dale
- Subjects
Genetics ,QH426-470 - Abstract
Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs.
- Published
- 2011
- Full Text
- View/download PDF
16. In vitropersistence level reflectsin vivoantibiotic survival of naturalPseudomonas aeruginosaisolates in a murine lung infection model
- Author
-
Laure Verstraete, Juliana Aizawa, Matthias Govaerts, Linda De Vooght, Jan Michiels, Bram Van den Bergh, and Paul Cos
- Abstract
Nowadays, clinicians are more and more confronted with the limitations of antibiotics to completely cure bacterial infections in patients. It has long been assumed that only antibiotic resistance plays a pivotal role in this. Indeed, the worldwide emergence of antibiotic resistance is considered as one of the major health threats of the 21stcentury. However, the presence of persister cells also has a significant influence on treatment outcomes. These antibiotic-tolerant cells are present in every bacterial population and are the result of the phenotypic switching of normal, antibiotic-sensitive cells. Persister cells complicate current antibiotic therapies and contribute to the development of resistance. In the past, extensive research has been performed to investigate persistence in laboratory settings, however, antibiotic tolerance in conditions that mimic the clinical setting is still poorly understood. In this study, we have optimized a mouse model for lung infections of the opportunistic pathogenPseudomonas aeruginosa. In this model, mice are intratracheally infected withP. aeruginosaembedded in seaweed alginate beads and subsequently treated with tobramycin via nasal droplets. A strain panel of 18P. aeruginosaisolates originating from environmental, human and animal clinical sources was selected to assess survival in the animal model. These survival levels were positively correlated with the survival levels determined via time-kill assays which is a common method to study persistence in the laboratory. We showed that both survival levels are comparable and thus that the classical persister assays are indicative for antibiotic tolerance in a clinical setting. The optimized animal model also allows us to test potential antipersister molecules and study persistence.ImportanceThe importance of targeting persister cells in antibiotic therapies becomes more evident as these antibiotic-tolerant cells underlie relapsing infections and resistance development. Here, we studied persistence in a clinically relevant pathogen,Pseudomonas aeruginosa. It is one of the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterobacterspp.) that are considered as a major health threat.P. aeruginosais mostly known for causing chronic lung infections in cystic fibrosis patients. We mimicked these lung infections in a mouse model to study persistence in more clinical conditions. We showed that the survival levels of naturalP. aeruginosaisolates in this model are positively correlated with the survival levels measured in classical persistence assays. These results not only validate the use of our current techniques to study persistence, but also open opportunities to study new persistence mechanisms or evaluate new antipersister compoundsin vivo.
- Published
- 2022
- Full Text
- View/download PDF
17. Antibiotic-tolerant persisters are pervasive among clinical Streptococcus pneumoniae isolates and show strong condition-dependence
- Author
-
Nele Geerts, Linda De Vooght, Ioannis Passaris, Bram Van den Bergh, and Paul Cos
- Abstract
Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet, in S. pneumoniae nothing is known about antibiotic-tolerant persisters. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. Here, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. In optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which shows that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof-of-concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections which will set stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research.IMPORTANCES. pneumoniae is considered a serious threat by the Centers of Disease Control and Prevention through arising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance and more specifically by antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting persistence is a general trait in S. pneumoniae cultures and that a broad range of genetic elements are controlling the phenotype. Together, our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.
- Published
- 2022
- Full Text
- View/download PDF
18. Targeting the tsetse-trypanosome interplay using genetically engineered Sodalis glossinidius
- Author
-
Linda De Vooght, Karin De Ridder, Shahid Hussain, Benoît Stijlemans, Patrick De Baetselier, Guy Caljon, Jan Van Den Abbeele, Department of Bio-engineering Sciences, Faculty of Sciences and Bioengineering Sciences, Cellular and Molecular Immunology, and Vriendenkring VUB
- Subjects
Trypanosoma ,Tsetse Flies ,fungi ,Immunology ,Trypanosoma brucei brucei ,Single-Domain Antibodies ,Microbiology ,Enterobacteriaceae ,Virology ,parasitic diseases ,Genetics ,Animals ,Parasitology ,Human medicine ,Symbiosis ,Biology ,Molecular Biology - Abstract
Sodalis glossinidius, a secondary bacterial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components interfering with African trypanosome transmission (i.e. paratransgenesis). Nanobodies (Nbs) have been proposed as potential candidates to target the parasite during development in the tsetse fly. In this study, we have generated an immune Nb-library and developed a panning strategy to select Nbs against the Trypanosoma brucei brucei procyclic developmental stage present in the tsetse fly midgut. Selected Nbs were expressed, purified, assessed for binding and tested for their impact on the survival and growth of in vitro cultured procyclic T. b. brucei parasites. Next, we engineered S. glossinidius to express the selected Nbs and validated their ability to block T. brucei development in the tsetse fly midgut. Genetically engineered S. glossinidius expressing Nb_88 significantly compromised parasite development in the tsetse fly midgut both at the level of infection rate and parasite load. Interestingly, expression of Nb_19 by S. glossinidius resulted in a significantly enhanced midgut establishment. These data are the first to show in situ delivery by S. glossinidius of effector molecules that can target the trypanosome-tsetse fly crosstalk, interfering with parasite development in the fly. These proof-of-principle data represent a major step forward in the development of a control strategy based on paratransgenic tsetse flies. Finally, S. glossinidius-based Nb delivery can also be applied as a powerful laboratory tool to unravel the molecular determinants of the parasite-vector association.
- Published
- 2021
19. Innate immunity in the tsetse fly (Glossina), vector of African trypanosomes
- Author
-
Irina Matetovici, Linda De Vooght, and Jan Van Den Abbeele
- Subjects
0301 basic medicine ,Trypanosoma ,Tsetse Flies ,030106 microbiology ,Immunology ,Zoology ,03 medical and health sciences ,Immune system ,Immunity ,parasitic diseases ,medicine ,Animals ,Humans ,African trypanosomiasis ,Symbiosis ,Innate immune system ,Bacteria ,Obligate ,biology ,fungi ,Tsetse fly ,medicine.disease ,biology.organism_classification ,Immunity, Innate ,Insect Vectors ,Trypanosomiasis, African ,030104 developmental biology ,Vector (epidemiology) ,Host-Pathogen Interactions ,Trypanosomiasis ,Developmental Biology - Abstract
Tsetse flies (Glossina sp.) are medically and veterinary important vectors of African trypanosomes, protozoan parasites that cause devastating diseases in humans and livestock in sub-Saharan Africa. These flies feed exclusively on vertebrate blood and harbor a limited diversity of obligate and facultative bacterial commensals. They have a well-developed innate immune system that plays a key role in protecting the fly against invading pathogens and in modulating the fly's ability to transmit African trypanosomes. In this review, we briefly summarize our current knowledge on the tsetse fly innate immune system and its interaction with the bacterial commensals and the trypanosome parasite.
- Published
- 2019
- Full Text
- View/download PDF
20. Combining paratransgenesis with SIT: impact of ionizing radiation on the DNA copy number of Sodalis glossinidius in tsetse flies
- Author
-
Andrew G. Parker, Robert L. Mach, Jan Van Den Abbeele, Linda De Vooght, Güler Demirbas-Uzel, Adly M. M. Abd-Alla, and Marc J. B. Vreysen
- Subjects
Male ,0301 basic medicine ,Microbiology (medical) ,Sodalis ,food.ingredient ,Tsetse Flies ,030106 microbiology ,lcsh:QR1-502 ,Zoology ,Paratransgenesis ,Symbiont ,Insect Control ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,Sterile insect technique ,food ,Enterobacteriaceae ,Trypanosomiasis ,Radiation, Ionizing ,medicine ,Animals ,Symbiosis ,biology ,Trypanosomosis ,Research ,fungi ,Enterobacteriaceae Infections ,Sodalis glossinidius ,Tsetse fly ,DNA ,biology.organism_classification ,medicine.disease ,Glossinidae ,Insect Vectors ,Glossina morsitans morsitans ,030104 developmental biology ,Wigglesworthia ,Female ,Wolbachia - Abstract
Background Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the causative agents of African Trypanosomosis, which has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. The sterile insect technique (SIT) has shown to be a powerful method to manage tsetse fly populations when used in the frame of an area-wide integrated pest management (AW-IPM) program. To date, the release of sterile males to manage tsetse fly populations has only been implemented in areas to reduce transmission of animal African Trypanosomosis (AAT). The implementation of the SIT in areas with Human African Trypanosomosis (HAT) would require additional measures to eliminate the potential risk associated with the release of sterile males that require blood meals to survive and hence, might contribute to disease transmission. Paratransgenesis offers the potential to develop tsetse flies that are refractory to trypanosome infection by modifying their associated bacteria (Sodalis glossinidius) here after referred to as Sodalis. Here we assessed the feasibility of combining the paratransgenesis approach with SIT by analyzing the impact of ionizing radiation on the copy number of Sodalis and the vectorial capacity of sterilized tsetse males. Results Adult Glossina morsitans morsitans that emerged from puparia irradiated on day 22 post larviposition did not show a significant decline in Sodalis copy number as compared with non-irradiated flies. Conversely, the Sodalis copy number was significantly reduced in adults that emerged from puparia irradiated on day 29 post larviposition and in adults irradiated on day 7 post emergence. Moreover, irradiating 22-day old puparia reduced the copy number of Wolbachia and Wigglesworthia in emerged adults as compared with non-irradiated controls, but the radiation treatment had no significant impact on the vectorial competence of the flies. Conclusion Although the radiation treatment significantly reduced the copy number of some tsetse fly symbionts, the copy number of Sodalis recovered with time in flies irradiated as 22-day old puparia. This recovery offers the opportunity to combine a paratransgenesis approach – using modified Sodalis to produce males refractory to trypanosome infection – with the release of sterile males to minimize the risk of disease transmission, especially in HAT endemic areas. Moreover, irradiation did not increase the vector competence of the flies for trypanosomes. Electronic supplementary material The online version of this article (10.1186/s12866-018-1283-8) contains supplementary material, which is available to authorized users.
- Published
- 2018
- Full Text
- View/download PDF
21. Enhancing vector refractoriness to trypanosome infection : achievements, challenges and perspectives
- Author
-
Adly M. M. Abd-Alla, İkbal Agah İnce, Fathiya M. Khamis, Anne Geiger, Irene K. Meki, Imna I. Malele, Florence N. Wamwiri, Linda De Vooght, Flobert Njiokou, Daniela I. Schneider, Brian L. Weiss, Güler Demirbas-Uzel, Sørge Kelm, Just M. Vlak, Henry M. Kariithi, and Acibadem University Dspace
- Subjects
0301 basic medicine ,Microbiology (medical) ,Trypanosoma ,Glossina ,Tsetse Flies ,030106 microbiology ,Laboratory of Virology ,lcsh:QR1-502 ,Paratransgenesis ,Trypanosoma-refractoriness ,sterile insect technique ,Biology ,Microbiology ,Insect Control ,lcsh:Microbiology ,Laboratorium voor Virologie ,03 medical and health sciences ,Sterile insect technique ,Insect pest management ,Correspondence ,Trypanosoma-refractoriness, sterile insect technique ,Animals ,Hytrosaviridae ,Symbiosis ,Molecular interactions ,business.industry ,Microbiota ,Tsetse fly ,biology.organism_classification ,PE&RC ,Biotechnology ,Insect Vectors ,Trypanosomiasis, African ,Vector (epidemiology) ,Neglected tropical diseases ,Female ,business ,Disease transmission ,Vector competence - Abstract
With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013–2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP’s major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.
- Published
- 2018
22. Paternal Transmission of a Secondary Symbiont during Mating in the Viviparous Tsetse Fly
- Author
-
Jan Van Den Abbeele, Linda De Vooght, Jos Van Hees, and Guy Caljon
- Subjects
Male ,Genome evolution ,Sodalis ,food.ingredient ,HOST ,Glossina ,APHIDS ,Gene Transfer, Horizontal ,Tsetse Flies ,Evolution, Molecular ,food ,SODALIS-GLOSSINIDIUS ,Enterobacteriaceae ,Genetics ,Animals ,Mating ,Symbiosis ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Coevolution ,Discoveries ,biology ,Host (biology) ,Ecology ,Reproduction ,fungi ,Sodalis glossinidius ,Tsetse fly ,Biology and Life Sciences ,genetic diversity ,biology.organism_classification ,symbiont ,Evolutionary biology ,paternal transmission ,Female ,Horizontal transmission ,Genome, Bacterial - Abstract
Sodalis glossinidius, a maternally inherited secondary symbiont of the tsetse fly, is a bacterium in the early/intermediate state of the transition toward symbiosis, representing an important model for investigating establishment and evolution of insect-bacteria symbiosis. The absence of phylogenetic congruence in tsetse-Sodalis coevolution and the existence of Sodalis genotypic diversity in field flies are suggestive for a horizontal transmission route. However, to date no natural mechanism for the horizontal transfer of this symbiont has been identified. Using novel methodologies for the stable fluorescent-labeling and introduction of modified Sodalis in tsetse flies, we unambiguously show that male-borne Sodalis is 1) horizontally transferred to females during mating and 2) subsequently vertically transmitted to the progeny, that is, paternal transmission. This mixed mode of transmission has major consequences regarding Sodalis' genome evolution as it can lead to coinfections creating opportunities for lateral gene transfer which in turn could affect the interaction with the tsetse host.
- Published
- 2015
23. Delivery of a functional anti-trypanosome Nanobody in different tsetse fly tissues via a bacterial symbiont, Sodalis glossinidius
- Author
-
Karina De Ridder, Jan Van Den Abbeele, Guy Caljon, and Linda De Vooght
- Subjects
EXPRESSION ,Trypanosoma ,Sodalis ,food.ingredient ,Glossina ,Tsetse Flies ,Population ,MOSQUITOS ,Antibodies, Protozoan ,Gene Expression ,Bioengineering ,Paratransgenesis ,Symbiont ,(3-10) ,Applied Microbiology and Biotechnology ,Microbiology ,food ,MALARIA ,Enterobacteriaceae ,Midgut ,In vivo ,Animals ,education ,Symbiosis ,education.field_of_study ,Recombinant ,biology ,Functional ,Host (biology) ,Effector ,Research ,fungi ,Sodalis glossinidius ,Tsetse fly ,Biology and Life Sciences ,Single-Domain Antibodies ,biology.organism_classification ,Insect Vectors ,(3–10) ,Nanobody ,Delivery ,Biotechnology ,Symbiotic bacteria - Abstract
Background Sodalis glossinidius, a vertically transmitted microbial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components that reduce or eliminate the capability of the tsetse fly host to transmit parasitic trypanosomes, an approach also known as paratransgenesis. An essential step in developing paratransgenic tsetse is the stable colonization of adult flies and their progeny with recombinant Sodalis bacteria, expressing trypanocidal effector molecules in tissues where the parasite resides. Results In this study, Sodalis was tested for its ability to deliver functional anti-trypanosome nanobodies (Nbs) in Glossina morsitans morsitans. We characterized the in vitro and in vivo stability of recombinant Sodalis (recSodalis) expressing a potent trypanolytic nanobody, i.e. Nb_An46. We show that recSodalis is competitive with WT Sodalis in in vivo conditions and that tsetse flies transiently cleared of their endogenous WT Sodalis population can be successfully repopulated with recSodalis at high densities. In addition, vertical transmission to the offspring was observed. Finally, we demonstrated that recSodalis expressed significant levels (ng range) of functional Nb_An46 in different tsetse fly tissues, including the midgut where an important developmental stage of the trypanosome parasite occurs. Conclusions We demonstrated the proof-of-concept that the Sodalis symbiont can be genetically engineered to express and release significant amounts of functional anti-trypanosome Nbs in different tissues of the tsetse fly. The application of this innovative concept of using pathogen-targeting nanobodies delivered by insect symbiotic bacteria could be extended to other vector-pathogen systems. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0156-6) contains supplementary material, which is available to authorized users.
- Published
- 2014
- Full Text
- View/download PDF
24. Options for the delivery of anti-pathogen molecules in arthropod vectors
- Author
-
Jan Van Den Abbeele, Guy Caljon, Linda De Vooght, and Cellular and Molecular Immunology
- Subjects
trypanosomiasis ,Computational biology ,Animals, Genetically Modified ,03 medical and health sciences ,Immune system ,Animals ,Pest Control, Biological ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,anti-pathogen molecules ,0303 health sciences ,Innate immune system ,biology ,(para)transgenesis ,030306 microbiology ,Effector ,Tsetse fly ,Arthropod Vectors ,biochemical phenomena, metabolism, and nutrition ,Single-Domain Antibodies ,Acquired immune system ,biology.organism_classification ,Virology ,Vector (epidemiology) ,biology.protein ,Nanobody ,Arthropod ,Vector borne disease ,Antibody ,Arthropod Vector - Abstract
Blood feeding arthropods are responsible for the transmission of a large array of medically important infectious agents that include viruses, bacteria, protozoan parasites and helminths. The recent development of transgenic and paratransgenic technologies have enabled supplementing the immune system of these arthropod vectors with anti-pathogen effector molecules in view of compromising their vector competence for these microbial agents. The characteristics of the selected anti-pathogen compound will largely determine the efficacy and specificity of this approach. Low specificity will generally result in bystander effects, likely having a direct or indirect fitness cost for the arthropod. In contrast, the use of highly specific compounds from the adaptive immune system of vertebrates such as antibody derived fragments is more likely to enable highly specific effects without conferring a selective disadvantage to the (para)transgenic arthropods. Here, Nanobodies® are excellent candidates to increase the immune competence of arthropods. Moreover they were shown to exert a novel type of anti-pathogen activity that uniquely depends on their small size.
- Published
- 2012
25. Attenuation of the Sensing Capabilities of PhoQ in Transition to Obligate Insect–Bacterial Association
- Author
-
Colin Dale, Jan Van Den Abbeele, Kari Smith, Mauricio H. Pontes, and Linda De Vooght
- Subjects
Cancer Research ,Applied Microbiology ,Regulator ,Gene Expression ,medicine.disease_cause ,Gene expression ,Magnesium ,Promoter Regions, Genetic ,Genetics (clinical) ,Adenosine Triphosphatases ,Genetics ,0303 health sciences ,Mutation ,biology ,Sodalis glossinidius ,Salmonella enterica ,Biological Evolution ,Lipid A ,Research Article ,Transcriptional Activation ,lcsh:QH426-470 ,Tsetse Flies ,Molecular Sequence Data ,Virulence ,Microbiology ,Host Specificity ,Molecular Genetics ,03 medical and health sciences ,Bacterial Proteins ,Enterobacteriaceae ,Escherichia coli ,medicine ,Animals ,Secretion ,Symbiosis ,Biology ,Molecular Biology ,Gene ,Alleles ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Evolutionary Biology ,Base Sequence ,030306 microbiology ,Membrane Transport Proteins ,Gene Expression Regulation, Bacterial ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,lcsh:Genetics ,Response regulator ,Microbial Evolution ,Gene Function ,Antimicrobial Cationic Peptides - Abstract
Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs., Author Summary Mutualistic insect endosymbionts are known to undergo a process of degenerative evolution that streamlines their gene inventory in accordance with the obligate nature of the host associated lifestyle. Here we show that the mutualistic insect endosymbiont Sodalis glossinidius utilizes a two-component regulatory system (PhoP-PhoQ) that evolved to facilitate the regulation of virulence genes in facultative pathogens in accordance with environmental magnesium. While the regulatory targets of PhoP-PhoQ are conserved among S. glossinidius and a wide range of bacterial pathogens, the S. glossinidius PhoP-PhoQ system is unusual because it has a substantially reduced ability to sense magnesium as a repressing signal. This enables S. glossinidius to express genes encoding critical symbiosis determinants within host environments that are rich in both magnesium and antimicrobial peptides. These findings illustrate how a change in the ecology of a host–symbiont association can influence the dynamic functionality of a regulatory system in the symbiont.
- Published
- 2011
- Full Text
- View/download PDF
26. Expression and extracellular release of a functional anti-trypanosome Nanobody® in Sodalis glossinidius, a bacterial symbiont of the tsetse fly
- Author
-
Linda De Vooght, Benoit Stijlemans, Jan Van Den Abbeele, Patrick De Baetselier, Marc Coosemans, Guy Caljon, and Cellular and Molecular Immunology
- Subjects
Signal peptide ,Sodalis ,food.ingredient ,Glossina ,Tsetse Flies ,Recombinant Fusion Proteins ,Trypanosoma brucei brucei ,lcsh:QR1-502 ,Protozoan Proteins ,Antibodies, Protozoan ,Gene Expression ,Paratransgenesis ,Expression ,Bioengineering ,Protein Sorting Signals ,Trypanosoma brucei ,Applied Microbiology and Biotechnology ,lcsh:Microbiology ,Microbiology ,paratransgenesis ,03 medical and health sciences ,food ,Enterobacteriaceae ,Extracellular ,Animals ,Sodalis glossinidius ,Symbiosis ,Glycoproteins ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Effector ,Research ,Periplasmic space ,biology.organism_classification ,symbiont ,functional ,Periplasm ,Nanobody ,Biotechnology - Abstract
Background Sodalis glossinidius, a gram-negative bacterial endosymbiont of the tsetse fly, has been proposed as a potential in vivo drug delivery vehicle to control trypanosome parasite development in the fly, an approach known as paratransgenesis. Despite this interest of S. glossinidius as a paratransgenic platform organism in tsetse flies, few potential effector molecules have been identified so far and to date none of these molecules have been successfully expressed in this bacterium. Results In this study, S. glossinidius was transformed to express a single domain antibody, (Nanobody®) Nb_An33, that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of the parasite Trypanosoma brucei. Next, we analyzed the capability of two predicted secretion signals to direct the extracellular delivery of significant levels of active Nb_An33. We show that the pelB leader peptide was successful in directing the export of fully functional Nb_An33 to the periplasm of S. glossinidius resulting in significant levels of extracellular release. Finally, S. glossinidius expressing pelBNb_An33 exhibited no significant reduction in terms of fitness, determined by in vitro growth kinetics, compared to the wild-type strain. Conclusions These data are the first demonstration of the expression and extracellular release of functional trypanosome-interfering Nanobodies® in S. glossinidius. Furthermore, Sodalis strains that efficiently released the effector protein were not affected in their growth, suggesting that they may be competitive with endogenous microbiota in the midgut environment of the tsetse fly. Collectively, these data reinforce the notion for the potential of S. glossinidius to be developed into a paratransgenic platform organism.
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.