scholarly journals Dictyostelium discoideum as a Model System for Identification of Burkholderia pseudomallei Virulence Factors

2011 ◽  
Vol 79 (5) ◽  
pp. 2079-2088 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Maharsh K. Patel ◽  
Mark A. Schell
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Burkholderia Pseudomallei ◽  
Bacterial Pathogen ◽  
Model System ◽  
Virulence Determinants ◽  
Content Type ◽  
Throughput Model ◽  
Human Infections ◽  
High Throughput Assay

ABSTRACTBurkholderia pseudomalleiis an emerging bacterial pathogen and category B biothreat. Human infections withB. pseudomallei(called melioidosis) present as a range of manifestations, including acute septicemia and pneumonia. Although melioidosis can be fatal, little is known about the molecular basis ofB. pseudomalleipathogenicity, in part because of the lack of simple, genetically tractable eukaryotic models to facilitateen masseidentification of virulence determinants or explore host-pathogen interactions. Two assays, one high-throughput and one quantitative, were developed to monitor levels of resistance ofB. pseudomalleiand the closely related nearly avirulent speciesBurkholderia thailandensisto predation by the phagocytic amoebaDictyostelium discoideum. The quantitative assay showed that levels of resistance to, and survival within, amoeba by these bacteria and their known virulence mutants correlate well with their published levels of virulence in animals. Using the high-throughput assay, we screened a 1,500-memberB. thailandensistransposon mutant library and identified 13 genes involved in resistance to predation byD. discoideum. Orthologs of these genes were disrupted inB. pseudomallei, and nearly all mutants had similarly decreased resistance to predation byD. discoideum. For some mutants, decreased resistance also correlated with reduced survival in and cytotoxicity toward macrophages, as well as attenuated virulence in mice. These observations suggest that some factors required byB. pseudomalleifor resistance to environmental phagocytes also aid in resistance to phagocytic immune cells and contribute to disease in animals. Thus,D. discoideumprovides a novel, high-throughput model system for facilitating inquiry intoB. pseudomalleivirulence.

scholarly journals Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1Streptococcus pyogenes

2018 ◽  
Vol 200 (8) ◽  
Author(s):  
Stephan Brouwer ◽  
Amanda J. Cork ◽  
Cheryl-Lynn Y. Ong ◽  
Timothy C. Barnett ◽  
Nicholas P. West ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Phase ◽  
Cysteine Protease ◽  
Genetic Regulation ◽  
Bacterial Pathogen ◽  
Targeted Mutagenesis ◽  
Human Neutrophils ◽  
Content Type ◽  
Wide Range ◽  
Human Infections

ABSTRACTStreptococcus pyogenes(group AStreptococcus[GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation ofspeBexpression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction ofspeBgene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448ΔpepOmutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCEDespite the continuing susceptibility ofS. pyogenesto penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that controlspeBgene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator ofspeBgene expression in the globally disseminated M1T1 clone and as being essential for virulence.

scholarly journals Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Alessandra Vitale ◽  
Sarah Paszti ◽  
Kohei Takahashi ◽  
Masanori Toyofuku ◽  
Gabriella Pessi ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Terminal Electron Acceptor ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Sufficient Energy ◽  
Genes Encoding ◽  
Human Infections

ABSTRACT Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis. Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc1, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N2O, which may play a role in the dispersal of B. thailandensis biofilms. IMPORTANCE Burkholderia thailandensis is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen Burkholderia pseudomallei, the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of B. pseudomallei is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of N2O to N2, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that N2O may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.

scholarly journals Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Michelle K. Paczosa ◽  
Rebecca J. Silver ◽  
Anne L. McCabe ◽  
Albert K. Tai ◽  
Colin H. McLeish ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bacterial Pathogen ◽  
Reactive Oxygen ◽  
Antimicrobial Activities ◽  
Virulence Determinants ◽  
Nitrogen Species ◽  
Content Type ◽  
Transposon Insertion ◽  
Tract Infections

ABSTRACT Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.

scholarly journals Luciferase-Based, High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes

2016 ◽  
Vol 60 (4) ◽  
pp. 2097-2107 ◽  
Author(s):  
Leonardo Lucantoni ◽  
David A. Fidock ◽  
Vicky M. Avery
Keyword(s):  
Plasmodium Falciparum ◽  
High Throughput ◽  
Early Stage ◽  
Stage Iv ◽  
Cost Effective ◽  
Luciferase Assay ◽  
Content Type ◽  
Starting Point ◽  
Gene Assay ◽  
High Throughput Assay

ABSTRACTThe discovery of new antimalarial drugs able to target both the asexual and gametocyte stages ofPlasmodium falciparumis critical to the success of the malaria eradication campaign. We have developed and validated a robust, rapid, and cost-effective high-throughput reporter gene assay to identify compounds active against late-stage (stage IV and V) gametocytes. The assay, which is suitable for testing compound activity at incubation times up to 72 h, demonstrates excellent quality and reproducibility, with averageZ′ values of 0.85 ± 0.01. We used the assay to screen more than 10,000 compounds from three chemically diverse libraries. The screening outcomes highlighted the opportunity to use collections of compounds with known activity against the asexual stages of the parasites as a starting point for gametocytocidal activity detection in order to maximize the chances of identifying gametocytocidal compounds. This assay extends the capabilities of our previously reported luciferase assay, which tested compounds against early-stage gametocytes, and opens possibilities to profile the activities of gametocytocidal compounds over the entire course of gametocytogenesis.

scholarly journals A novel FK-506-binding-like protein that lacks peptidyl-prolyl isomerase activity is involved in intracellular infection and in vivo virulence of Burkholderia pseudomallei

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2629-2638 ◽  
Author(s):  
Isobel H. Norville ◽  
Katrin Breitbach ◽  
Kristin Eske-Pogodda ◽  
Nicholas J. Harmer ◽  
Mitali Sarkar-Tyson ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Pathogenic Bacteria ◽  
Plaque Assay ◽  
Bacterial Pathogen ◽  
Virulence Determinants ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Fk 506 ◽  
Peptidyl Prolyl Isomerase ◽  
Intracellular Infection

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen causing melioidosis, an often fatal infectious disease that is endemic in several tropical and subtropical areas around the world. We previously described a Ptk2 cell-based plaque assay screening system of B. pseudomallei transposon mutants that led to the identification of several novel virulence determinants. Using this approach we identified a mutant with reduced plaque formation in which the BPSL0918 gene was disrupted. BPSL0918 encodes a putative FK-506-binding protein (FKBP) representing a family of proteins that typically possess peptidyl-prolyl isomerase (PPIase) activity. A B. pseudomallei ΔBPSL0918 mutant showed a severely impaired ability to resist intracellular killing and to replicate within primary macrophages. Complementation of the mutant fully restored its ability to grow intracellularly. Moreover, B. pseudomallei ΔBPSL0918 was significantly attenuated in a murine model of infection. Structural modelling confirmed a modified FKBP fold of the BPSL0918-encoded protein but unlike virulence-associated FKBPs from other pathogenic bacteria, recombinant BPSL0918 protein did not possess PPIase activity in vitro. In accordance with this observation BPSL0918 exhibits several mutations in residues that have been proposed to mediate PPIase activity in other FKBPs. To our knowledge this B. pseudomallei FKBP represents the first example of this protein family which lacks PPIase activity but is important in intracellular infection of a bacterial pathogen.

scholarly journals Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

2014 ◽  
Vol 21 (5) ◽  
pp. 689-697 ◽  
Author(s):  
Jinfu Xie ◽  
Melanie Horton ◽  
Julie Zorman ◽  
Joseph M. Antonello ◽  
Yuhua Zhang ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
High Throughput ◽  
Neutralizing Antibodies ◽  
Severe Disease ◽  
Molar Ratio ◽  
Seeding Density ◽  
Binary Toxin ◽  
Hyperimmune Serum ◽  
Content Type ◽  
High Throughput Assay

ABSTRACTClostridium difficilestrains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence ofC. difficileinfection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized withC. difficilebinary toxin-containing vaccines. The assay will be useful forC. difficilediagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.

scholarly journals A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Terrence Cheng ◽  
Nelson S. Torres ◽  
Ping Chen ◽  
Anand Srinivasan ◽  
Sandra Cardona ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
High Throughput ◽  
Three Dimensional ◽  
Hanging Drop ◽  
Methicillin Resistant ◽  
Content Type ◽  
Throughput Model

ABSTRACT Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection. IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

scholarly journals Peptidyl-Prolyl Isomerase ppiB Is Essential for Proteome Homeostasis and Virulence in Burkholderia pseudomallei

2019 ◽  
Vol 87 (10) ◽  
Author(s):  
Nicole M. Bzdyl ◽  
Nichollas E. Scott ◽  
Isobel H. Norville ◽  
Andrew E. Scott ◽  
Timothy Atkins ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Antimicrobial Treatment ◽  
Infection Model ◽  
Virulence Determinants ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Cyclophilin B ◽  
Novel Treatments

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes. Cyclophilins catalyze the cis-trans isomerization of xaa-proline bonds, a rate-limiting step in protein folding which has been shown to be important for bacterial virulence. B. pseudomallei carries a putative cyclophilin B gene, ppiB, the role of which was investigated. A B. pseudomallei ΔppiB (BpsΔppiB) mutant strain demonstrates impaired biofilm formation and reduced motility. Macrophage invasion and survival assays showed that although the BpsΔppiB strain retained the ability to infect macrophages, it had reduced survival and lacked the ability to spread cell to cell, indicating ppiB is essential for B. pseudomallei virulence. This is reflected in the BALB/c mouse infection model, demonstrating the requirement of ppiB for in vivo disease dissemination and progression. Proteomic analysis demonstrates that the loss of PpiB leads to pleiotropic effects, supporting the role of PpiB in maintaining proteome homeostasis. The loss of PpiB leads to decreased abundance of multiple virulence determinants, including flagellar machinery and alterations in type VI secretion system proteins. In addition, the loss of ppiB leads to increased sensitivity toward multiple antibiotics, including meropenem and doxycycline, highlighting ppiB inhibition as a promising antivirulence target to both treat B. pseudomallei infections and increase antibiotic efficacy.

scholarly journals Regulation of Type VI Secretion System during Burkholderia pseudomallei Infection

2011 ◽  
Vol 79 (8) ◽  
pp. 3064-3073 ◽  
Author(s):  
Yahua Chen ◽  
Jocelyn Wong ◽  
Guang Wen Sun ◽  
Yichun Liu ◽  
Gek-Yen Gladys Tan ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Host Cells ◽  
Virulence Determinants ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Content Type ◽  
Regulatory Cascade ◽  
Type Vi Secretion ◽  
Absolute Dependence

ABSTRACTType III and type VI secretion systems (T3SSs and T6SSs, respectively) are critical virulence determinants in several Gram-negative pathogens. InBurkholderia pseudomallei, the T3SS-3 and T6SS-1 clusters have been implicated in bacterial virulence in mammalian hosts. We recently discovered a regulatory cascade that coordinately controls the expression of T3SS-3 and T6SS-1. BsaN is a central regulator located within T3SS-3 for the expression of T3SS-3 effectors and regulators for T6SS-1 such as VirA-VirG (VirAG) and BprC. Whereas T6SS-1 gene expression was completely dependent on BprC when bacteria were grown in medium, the expression inside host cells was dependent on the two-component sensor-regulator VirAG, with the exception of thetssABoperon, which was dependent primarily on BprC. VirAG and BprC initiate different transcriptional start sites within T6SS-1, and VirAG is able to activate thehcp1promoter directly. We also provided novel evidence thatvirAG,bprC, andtssABare critical for T6SS-1 function in macrophages. Furthermore,virAGandbprCregulator mutants were avirulent in mice, demonstrating the absolute dependence of T6SS-1 expression on these regulatorsin vivo.

scholarly journals Protein Antigens Increase the Protective Efficacy of a Capsule-Based Vaccine against Staphylococcus aureus in a Rat Model of Osteomyelitis

2013 ◽  
Vol 82 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Santiago M. Lattar ◽  
Mariángeles Noto Llana ◽  
Philippe Denoël ◽  
Sophie Germain ◽  
Fernanda R. Buzzola ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Conjugate Vaccine ◽  
Rat Model ◽  
Morphological Changes ◽  
Protective Efficacy ◽  
Bacterial Load ◽  
Bacterial Pathogen ◽  
Protein Antigens ◽  
Content Type ◽  
Human Infections

ABSTRACTStaphylococcus aureusis an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-componentS. aureusvaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing bothS. aureuscapsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy againstS. aureusosteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease.

Sign in / Sign up

Export Citation Format

Share Document