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 Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

scholarly journals Mutations That Impact the Enteropathogenic Escherichia coli Cpx Envelope Stress Response Attenuate Virulence in Galleria mellonella

2012 ◽  
Vol 80 (9) ◽  
pp. 3077-3085 ◽  
Author(s):  
S. Leuko ◽  
T. L. Raivio
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Virulence Determinants ◽  
Content Type ◽  
Type Iii ◽  
Envelope Stress

ABSTRACTIn this paper, we show that the larvae of the greater wax moth,Galleria mellonella, can be used as a model to study enteropathogenicEscherichia coli(EPEC) virulence.G. mellonellalarvae are killed after infection with EPEC type strain E2348/69 but not by an attenuated derivative that expresses diminished levels of the major virulence determinants or by a mutant specifically defective in type III secretion (T3S). Infecting EPEC inhabit the larval hemocoel only briefly and then become localized to melanized capsules, where they remain extracellular. Previously, it was shown that mutations affecting the Cpx envelope stress response lead to diminished expression of the bundle-forming pilus (BFP) and the type III secretion system (T3SS). We demonstrate that mutations that activate the Cpx pathway have a dramatic effect on the ability of the bacterium to establish a lethal infection, and this is correlated with an inability to growin vivo. Infection with allE. colistrains led to increased expression of the antimicrobial peptides (AMPs) gloverin and cecropin, although strain- and AMP-specific differences were observed, suggesting that theG. mellonellahost perceives attenuated strains and Cpx mutants in unique manners. Overall, this study shows thatG. mellonellais an economical, alternative infection model for the preliminary study of EPEC host-pathogen interactions, and that induction of the Cpx envelope stress response leads to defects in virulence.

scholarly journals Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

2015 ◽  
Vol 14 (8) ◽  
pp. 834-844 ◽  
Author(s):  
Ranjith Rajendran ◽  
Elisa Borghi ◽  
Monica Falleni ◽  
Federica Perdoni ◽  
Delfina Tosi ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Fungal Infections ◽  
Infection Model ◽  
Content Type

ABSTRACT Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo . In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

scholarly journals Orientia tsutsugamushiIs Highly Susceptible to the RNA Polymerase Switch Region Inhibitor Corallopyronin AIn VitroandIn Vivo

2018 ◽  
Vol 62 (4) ◽  
pp. e01732-17 ◽  
Author(s):  
Fredericke Kock ◽  
Matthias Hauptmann ◽  
Anke Osterloh ◽  
Till F. Schäberle ◽  
Sven Poppert ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Scrub Typhus ◽  
Adaptive Immune Response ◽  
Antimicrobial Treatment ◽  
Infection Model ◽  
Switch Region ◽  
Content Type ◽  
Mouse Infection Model

ABSTRACTScrub typhus is a potentially lethal infection caused by the obligate intracellular bacteriumOrientia tsutsugamushi. Reports on the emergence of doxycycline-resistant strains highlight the urgent need to develop novel antiinfectives against scrub typhus. Corallopyronin A (CorA) is a novel α-pyrone compound synthesized by the myxobacteriumCorallococcus coralloidesthat was characterized as a noncompetitive inhibitor of the switch region of the bacterial RNA polymerase (RNAP). We investigated the antimicrobial action of CorA against the human-pathogenic Karp strain ofO. tsutsugamushiin vitroandin vivo. The MIC of CorA againstO. tsutsugamushiwas remarkably low (0.0078 μg/ml), 16-fold lower than that againstRickettsia typhi. In the lethal intraperitonealO. tsutsugamushimouse infection model, a minimum daily dose of 100 μg CorA protected 100% of infected mice. Two days of treatment were sufficient to confer protection. In contrast to BALB/c mice, SCID mice succumbed to the infection despite treatment with CorA or tetracycline, suggesting that antimicrobial treatment required synergistic action of the adaptive immune response. Similar to tetracycline, CorA did not prevent latent infection ofO. tsutsugamushiin vivo. However, latency was not caused by acquisition of antimicrobial resistance, sinceO. tsutsugamushireisolated from latently infected BALB/c mice remained fully susceptible to CorA. No mutations were found in the CorA-binding regions of the β and β′ RNAP subunit genesrpoBandrpoC. Inhibition of the RNAP switch region ofO. tsutsugamushiby CorA is therefore a novel and highly potent target for antimicrobial therapy for scrub typhus.

scholarly journals Type VI secretion system and its effectors PdpC, PdpD and OpiA contribute to Francisella virulence in Galleria mellonella larvae

2021 ◽  
Author(s):  
Maj Brodmann ◽  
Sophie Schnider ◽  
Marek Basler
Keyword(s):  
Galleria Mellonella ◽  
Response Regulator ◽  
Infection Model ◽  
Type Vi Secretion System ◽  
Infectious Dose ◽  
Infection Models

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a non-canonical T6SS, in an arthropod in vivo infection model is missing. Here we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of PdpC, PdpD or OpiA T6SS effectors is sufficient to kill G. mellonella larvae while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK and OpiB1-3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into function of T6SS and pathogenesis of Francisella.

scholarly journals Bacterial T6SS Effector EvpP Inhibits Neutrophil Recruitment via Jnk-Caspy Inflammasome Signaling In vivo

2018 ◽  
Author(s):  
Jinchao Tan ◽  
Dahai Yang ◽  
Zhuang Wang ◽  
Xin Zheng ◽  
Yuanxing Zhang ◽  
...  
Keyword(s):  
Bacterial Colonization ◽  
Critical Role ◽  
Neutrophil Recruitment ◽  
Host Cells ◽  
Infection Model ◽  
Inflammasome Activation ◽  
Type Vi Secretion System ◽  
Zebrafish Larvae

ABSTRACTThe type VI secretion system (T6SS) comprises dynamic complex bacterial contractile nanomachines and is used by many bacteria to inhibit or kill other prokaryotic or eukaryotic cells. Previous studies have revealed that T6SS is constitutively active in response to various stimuli, or fires effectors into host cells during infection. It has been proposed that the T6SS effector EvpP in Edwardsiella piscicida can inhibit NLRP3 inflammasome activation via the Ca2+-dependent JNK pathways. Here, we developed an in vivo infection model by microinjecting bacteria into the tail vein muscle of 3-day-post-fertilized zebrafish larvae, and found that both macrophages and neutrophils are essential for bacterial clearance. Further study revealed that EvpP plays a critical role in promoting the pathogenesis of E. piscicida via inhibiting the phosphorylation of Jnk signaling to reduce the expression of cxcl8a, mmp13 and IL-1β in vivo. Subsequently, by utilizing Tg (mpo:eGFP+/+) zebrafish larvae for E. piscicida infection, we found that the EvpP-inhibited Jnk-caspy inflammasome signaling axis significantly suppressed the recruitment of neutrophils to infection sites, and the caspy‐ or IL-1β-MO knockdown larvae were more susceptible to infection and failed to restrict bacterial colonization in vivo.IMPORTANCEInnate immunity is regulated by phagocytic cells and is critical for host control of bacterial infection. In many bacteria, T6SSs can affect bacterial virulence in certain environments, but little is known about the mechanisms underlying T6SS regulation of innate immune responses during infection in vivo. Here, we investigated the role of an E. piscicida T6SS effector EvpP in manipulating the reaction of neutrophils in vivo. We show that EvpP inhibits the activation of Jnk-caspy inflammasome pathway in zebrafish larvae, and reveal that macrophages are essential for neutrophil recruitment in vivo. This interaction improves our understanding about the complex and contextual role of a bacterial T6SS effector in modulating the action of myeloid cells during infection, and offers new insights into the warfare between bacterial weapons and host immunological surveillance.

scholarly journals Characterization of the Burkholderia pseudomallei K96243 Capsular Polysaccharide I Coding Region

2012 ◽  
Vol 80 (3) ◽  
pp. 1209-1221 ◽  
Author(s):  
Jon Cuccui ◽  
Timothy S. Milne ◽  
Nicholas Harmer ◽  
Alison J. George ◽  
Sarah V. Harding ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Bioinformatic Analysis ◽  
Chromosome 1 ◽  
Infection Model ◽  
Coding Region ◽  
Content Type ◽  
Group 3

Burkholderia pseudomalleiis the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule inB. pseudomalleiis essential for virulence.B. pseudomalleicapsular polysaccharide (CPS) I comprises unbranchedmanno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivatinggmhA,wcbJ, andwcbNinB. pseudomalleiK96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with theB. pseudomalleiK96243 mutants lacking a functional copy of eithergmhAorwcbJwere afforded significant levels of protection against a wild-typeB. pseudomalleiK96243 challenge.

scholarly journals Reversible Daptomycin Tolerance of Adherent Staphylococci in an Implant Infection Model

2011 ◽  
Vol 55 (7) ◽  
pp. 3510-3516 ◽  
Author(s):  
Anne-K. John ◽  
Mathias Schmaler ◽  
Nina Khanna ◽  
Regine Landmann
Keyword(s):  
Treatment Failure ◽  
Mutant Strain ◽  
Calcium Ions ◽  
Biofilm Formation ◽  
Infection Model ◽  
Content Type ◽  
Wall Components

ABSTRACTDaptomycin (DAP) is bactericidal against methicillin-resistantStaphylococcus aureus(MRSA)in vitro, but it failed to eradicate MRSA in an experimental model of implant-associated infection. We therefore investigated various factors which could explain treatment failure by evaluating DAP activity, including the role of different cell wall components, adherence, biofilm, and calcium ions (Ca2+)in vitroandin vivo. In the tissue cage infection model, DAP was active only prophylactically and against low inocula. To identify the mechanisms of treatment failure, thein vitroactivity of DAP against planktonic and adherent growingS. aureusandS. epidermidismutants, differing in their capacity of biofilm formation and adherence, was determined. For planktonic staphylococci, the MIC was 0.625 μg/ml. For adherent staphylococci, DAP reduced biofilms at 30 μg/ml. However, it did not kill adherent bacteria up to 500 μg/ml, independent of biofilm biosynthesis (theicamutant strain), nuclease (thenuc1/nuc2mutant strain), LPXTG-anchored adhesin (thesrtAmutant strain), autolysin (theatlmutant strain), or alanyl-LTA (thedltAmutant strain). Resistance of adherent staphylococci was not due to mutations of adherent bacteria, since staphylococci became DAP susceptible after detachment. Phenotypic tolerance was not explained by inactivation of DAP or inability of initial Ca2+-DAP complex formation. However, the addition of up to 100 mg/liter (2.5 mmol/liter) Ca2+gradually improved bactericidal activity toward adherent staphylococciin vitroand increased the prevention rate in the cage model from 40% to 60%. In summary, adherent staphylococci are resistant to DAP killing unless Ca2+is supplemented to physiologic concentrations.

scholarly journals Role of Energy Sensor TlpD of Helicobacter pylori in Gerbil Colonization and Genome Analyses after Adaptation in the Gerbil

2013 ◽  
Vol 81 (10) ◽  
pp. 3534-3551 ◽  
Author(s):  
Wiebke Behrens ◽  
Tobias Schweinitzer ◽  
Joena Bal ◽  
Martina Dorsch ◽  
André Bleich ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Human Isolate ◽  
Infection Model ◽  
Energy Yield ◽  
Content Type ◽  
Full Characterization ◽  
H Pylori ◽  
The Impact

ABSTRACTHelicobacter pylorimaintains colonization in its human host using a limited set of taxis sensors. TlpD is a proposed energy taxis sensor ofH. pyloriand dominant under environmental conditions of low bacterial energy yield. We studied the impact ofH. pyloriTlpD on colonizationin vivousing a gerbil infection model which closely mimics the gastric physiology of humans. A gerbil-adaptedH. pyloristrain, HP87 P7, showed energy-dependent behavior, while its isogenictlpDmutant lost it. A TlpD-complemented strain regained the wild-type phenotype. Infection of gerbils with the complemented strain demonstrated that TlpD is important for persistent infection in the antrum and corpus and suggested a role of TlpD in horizontal navigation and persistent corpus colonization. As a part of the full characterization of the model and to gain insight into the genetic basis ofH. pyloriadaptation to the gerbil, we determined the complete genome sequences of the gerbil-adapted strain HP87 P7, two HP87 P7tlpDmutants before and after gerbil passage, and the original human isolate, HP87. The integrity of the genome, including that of a functionalcagpathogenicity island, was maintained after gerbil adaptation. Genetic and phenotypic differences between the strains were observed. Major differences between the gerbil-adapted strain and the human isolate emerged, including evidence of recent recombination. Passage of thetlpDmutant through the gerbil selected for gain-of-function variation in a fucosyltransferase gene,futC(HP0093). In conclusion, a gerbil-adaptedH. pyloristrain with a stable genome has helped to establish that TlpD has important functions for persistent colonization in the stomach.

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