scholarly journals In VivoAssessment of Growth and Virulence Gene Expression during Commensal and Pathogenic Lifestyles ofluxABCDE-Tagged Enterococcus faecalis Strains in Murine Gastrointestinal and Intravenous Infection Models

2013 ◽  
Vol 79 (13) ◽  
pp. 3986-3997 ◽  
Author(s):  
Sabina Leanti La Rosa ◽  
Pat G. Casey ◽  
Colin Hill ◽  
Dzung B. Diep ◽  
Ingolf F. Nes ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Expression Profiles ◽  
Systemic Infection ◽  
Intestinal Lumen ◽  
Infection Model ◽  
Dependent Manner ◽  
Virulence Determinants ◽  
Specific Expression ◽  
Content Type

ABSTRACTCytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulentEnterococcus faecalisstrains. In an effort to explore the expression profiles of these virulence traitsin vivo, we have employedE. faecalisvariants expressing theluxABCDEcassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections ofGalleria mellonellacaterpillars and mice. Systemic infection ofG. mellonellawith bioluminescence-taggedE. faecalisMMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R2> 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (109CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation ofE. faecalisvirulence determinants and to study the spatiotemporal course of infection in living animals in real time.

scholarly journals SlyA Is a Transcriptional Regulator Involved in the Virulence of Enterococcus faecalis

2011 ◽  
Vol 79 (7) ◽  
pp. 2638-2645 ◽  
Author(s):  
Charlotte Michaux ◽  
Maurizio Sanguinetti ◽  
Fany Reffuveille ◽  
Yanick Auffray ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Peritoneal Macrophages ◽  
Bacterial Virulence ◽  
Transcriptional Analysis ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Wild Type Parent

ABSTRACTPhylogenetic analysis of the crystal structure of theEnterococcus faecalisSlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator inE. faecalisbiology, we dissected the genetic organization of theslyA-EF_3001 locus and constructed aslyAdeletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyAmutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that theslyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyAmutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

scholarly journals Comparative Pathogenicity of United Kingdom Isolates of the Emerging Pathogen Candida auris and Other Key Pathogenic Candida Species

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Andrew M. Borman ◽  
Adrien Szekely ◽  
Elizabeth M. Johnson
Keyword(s):  
Invasive Candidiasis ◽  
Fungal Pathogen ◽  
Galleria Mellonella ◽  
Systemic Infection ◽  
Mortality Rates ◽  
Infection Model ◽  
Candida Auris ◽  
Deep Tissue ◽  
Content Type

ABSTRACT The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus. Candida auris, first described in 2009, has since emerged as an important, multidrug-resistant, nosocomial agent of candidemia, with large outbreaks reported worldwide and high mortality rates associated with therapeutic failure. The current study employed C. auris isolates from a variety of centers in the United Kingdom to evaluate the pathogenicity of this emerging pathogen compared to that of other common pathogenic yeast species in the invertebrate Galleria mellonella infection model. We showed that C. auris isolates differ in their growth characteristics in vitro, with a proportion of isolates failing to release daughter cells after budding, resulting in the formation of large aggregates of cells that cannot be physically disrupted. Our results also demonstrate strain-specific differences in the behavior of C. auris in G. mellonella, with the aggregate-forming isolates exhibiting significantly less pathogenicity than their nonaggregating counterparts. Importantly, the nonaggregating isolates exhibited pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus, despite the fact that C. auris isolates do not produce hyphae and produce only rudimentary pseudohyphae either in vitro or in G. mellonella. IMPORTANCE The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus.

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 Construction and Application of aluxABCDEReporter System for Real-Time Monitoring of Enterococcus faecalis Gene Expression and Growth

2012 ◽  
Vol 78 (19) ◽  
pp. 7003-7011 ◽  
Author(s):  
Sabina Leanti La Rosa ◽  
Dzung B. Diep ◽  
Ingolf F. Nes ◽  
Dag Anders Brede
Keyword(s):  
Real Time ◽  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Viable Cell ◽  
Model Systems ◽  
Infection Model ◽  
Broad Host Range ◽  
Reporter System ◽  
Content Type

ABSTRACTThe present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging ofEnterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains ofE. faecaliswere constructed. pSL101 harbors theluxABCDEoperon from pPL2luxand the pREG696 broad-host-range replicon andaxe-txetoxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R2> 0.98) with the viable-cell count. We employedlux-taggedE. faecalisstrains to monitor growth in real time in milk and urinein vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in theGalleria mellonellamodel system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI ofE. faecalisand an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.

scholarly journals The Campylobacter jejuni Ferric Uptake Regulator Promotes Acid Survival and Cross-Protection against Oxidative Stress

2016 ◽  
Vol 84 (5) ◽  
pp. 1287-1300 ◽  
Author(s):  
Momen Askoura ◽  
Sabina Sarvan ◽  
Jean-François Couture ◽  
Alain Stintzi
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Galleria Mellonella ◽  
Acid Stress ◽  
Cross Protection ◽  
Infection Model ◽  
Dependent Manner ◽  
Ferric Uptake Regulator ◽  
Content Type ◽  
Acidic Conditions

Campylobacter jejuniis a prevalent cause of bacterial gastroenteritis in humans worldwide. The mechanisms by whichC. jejunisurvives stomach acidity remain undefined. In the present study, we demonstrated that theC. jejuniferric uptake regulator (Fur) plays an important role inC. jejuniacid survival and acid-induced cross-protection against oxidative stress. AC. jejuniΔfurmutant was more sensitive to acid than the wild-type strain. Profiling of the acid stimulon of theC. jejuniΔfurmutant allowed us to uncover Fur-regulated genes under acidic conditions. In particular, Fur was found to upregulate genes involved in flagellar and cell envelope biogenesis upon acid stress, and mutants with deletions of these genes were found to be defective in surviving acid stress. Interestingly, prior acid exposure ofC. jejunicross-protected against oxidative stress in a catalase (KatA)- and Fur-dependent manner. Western blotting and reverse transcription-quantitative PCR revealed increased expression of KatA upon acid stress. Electrophoretic mobility shift assays (EMSAs) demonstrated that the binding affinity between Fur and thekatApromoter is reducedin vitrounder conditions of low pH, rationalizing the higher levels of expression ofkatAunder acidic conditions. Strikingly, the Δfurmutant exhibited reduced virulence in both human epithelial cells and theGalleria mellonellainfection model. Altogether, this is the first study showing that, in addition to its role in iron metabolism, Fur is an important regulator ofC. jejuniacid responses and this function cross-protects against oxidative stress. Moreover, our results clearly demonstrate Fur's important role inC. jejunipathogenesis.

scholarly journals The PolyamineN-Acetyltransferase-Like Enzyme PmvE Plays a Role in the Virulence of Enterococcus faecalis

2014 ◽  
Vol 83 (1) ◽  
pp. 364-371 ◽  
Author(s):  
Cecilia Martini ◽  
Charlotte Michaux ◽  
Francesca Bugli ◽  
Alessandro Arcovito ◽  
Federica Iavarone ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Direct Interaction ◽  
Transcriptional Regulator ◽  
Peritoneal Macrophages ◽  
Polyamine Metabolism ◽  
Infection Model ◽  
Content Type ◽  
Biochemical Assays

We previously showed that the mutant strain ofEnterococcus faecalislacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of theslyAoperon,ef_3001, renamedpmvE(forpolyaminemetabolism andvirulence ofE. faecalis). PmvE shares strong homologies withN1-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used anE. faecalisstrain carrying the recombinant plasmid pMSP3535-pmvE(V19/p3535-pmvE), which allows the induction ofpmvEby addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence ofE. faecalisin theGalleria mellonellainfection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses anN-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence ofE. faecalis, likely through its involvement in the polyamine metabolism.

scholarly journals Pharmacodynamics of Daptomycin against Enterococcus faecium and Enterococcus faecalis in the Murine Thigh Infection Model

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
James M. Kidd ◽  
Kamilia Abdelraouf ◽  
Tomefa E. Asempa ◽  
Romney M. Humphries ◽  
David P. Nicolau
Keyword(s):  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Hill Equation ◽  
Infection Model ◽  
Free Drug Concentration ◽  
Time Curve ◽  
Content Type ◽  
Concentration Time ◽  
The Hill ◽  
Susceptibility Breakpoint

ABSTRACT The Clinical and Laboratory Standards Institute (CLSI) daptomycin MIC susceptibility breakpoint for the treatment of enterococcal infections is ≤4 μg/ml. However, patients receiving daptomycin for the treatment of infections caused by enterococci with MICs of ≤4 μg/ml may experience treatment failures. We assessed the pharmacodynamics of daptomycin against enterococci in a neutropenic murine thigh infection model and determined the exposures necessary for bacteriostasis and a 1-log10-CFU reduction of Enterococcus faecalis and Enterococcus faecium. We further characterized daptomycin efficacy at clinically achievable exposures. Six E. faecium and 6 E. faecalis isolates (daptomycin MICs, 0.5 to 32 μg/ml) were studied. Daptomycin was administered at various doses over 24 h to achieve area under the free drug concentration-time curve-to-MIC ratios (fAUC0–24/MIC) ranging from 1 to 148. Daptomycin regimens that simulate mean human exposures following doses of 6, 8, and 10 mg/kg of body weight/day were also studied. Efficacy was assessed by the differences in the number of log10 CFU per thigh at 24 h. The Hill equation was used to estimate the fAUC0–24/MIC required to achieve bacteriostasis and a 1-log10-CFU reduction. For E. faecium, a 1-log10-CFU reduction required an fAUC0–24/MIC of 12.9 (R2 = 0.71). For E. faecalis, a 1-log10-CFU reduction was not achieved, while the fAUC0–24/MIC required for stasis was 7.2 (R2 = 0.8). With a human-simulated regimen of 6 mg/kg/day, a 1-log10-CFU reduction was observed in 3/3 E. faecium isolates with MICs of <4 μg/ml and 0/3 E. faecium isolates with MICs of ≥4 μg/ml; however, a 1-log10-CFU reduction was not achieved for any of the 6 E. faecalis isolates. These results, alongside clinical data, prompt a reevaluation of the current breakpoint.

scholarly journals Immunization with Pneumococcal Polysaccharide Serotype 3 and Lipopolysaccharide Modulates Lung and Liver Inflammation during a Virulent Streptococcus pneumoniae Infection in Mice

2013 ◽  
Vol 20 (5) ◽  
pp. 639-650 ◽  
Author(s):  
Katherine H. Restori ◽  
Mary J. Kennett ◽  
A. Catharine Ross
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Acute Phase ◽  
Acute Phase Proteins ◽  
Expression Profiles ◽  
Cytokine Gene Expression ◽  
Dependent Manner ◽  
Pneumonia Severity ◽  
Content Type ◽  
Amyloid A

ABSTRACTVaccination reduces morbidity and mortality from pneumonia, but its effect on the tissue-level response to infection is still poorly understood. We evaluated pneumonia disease progression, acute-phase response, and lung gene expression profiles in mice inoculated intranasally with virulent Gram-positiveStreptococcus pneumoniaeserotype 3 (ST 3) with and without prior immunization with pneumococcal polysaccharide ST 3 (PPS3) or after coimmunization with PPS3 and a low dose of lipopolysaccharide (PPS3+LPS). Pneumonia severity was assessed in the acute phase at 5, 12, 24 and 48 h postinoculation (p.i.) and in the resolution phase at 7 days p.i. Primary PPS3-specific antibody production was upregulated, and IgM binding to pneumococci increased in PPS3-immunized mice. Immunizations with PPS3 or PPS3+LPS decreased bacterial recovery in the lung and blood at 24 and 48 h and increased survival. Microarray analysis of whole-lung RNA revealed significant changes in the acute-phase protein serum amyloid A (SAA) levels between noninfected and infected mice, and these changes were attenuated by immunization. SAA transcripts were higher in the liver and lungs of infected controls, and SAA protein was elevated in serum but decreased in PPS3-immunized mice. Thus, during a virulent pneumonia infection, prior immunization with PPS3 in an IgM-dependent manner as well as immunization with PPS3+LPS attenuated pneumonia severity and promoted resolution of infection, concomitant with significant regulation of cytokine gene expression levels in the lungs and acute-phase proteins in the lungs, liver, and serum.

scholarly journals Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus

2021 ◽  
Vol 11 ◽  
Author(s):  
Guillaume Ménard ◽  
Astrid Rouillon ◽  
Gevorg Ghukasyan ◽  
Mathieu Emily ◽  
Brice Felden ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Expression Profiles ◽  
Bacterial Pathogens ◽  
Infection Model ◽  
Animal Testing ◽  
Easy Method ◽  
Larval Infection ◽  
Over Time

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

scholarly journals ThemtfATranscription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus fumigatus

2014 ◽  
Vol 13 (6) ◽  
pp. 766-775 ◽  
Author(s):  
Timothy D. Smith ◽  
Ana M. Calvo
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Galleria Mellonella ◽  
Molecular Genetic ◽  
Colony Growth ◽  
Infection Model ◽  
Slight Reduction ◽  
Wild Type ◽  
Content Type ◽  
Factor Gene

ABSTRACTAspergillus fumigatusis the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies inA. fumigatuscould provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor genemtfAinA. fumigatus. Our results revealed thatmtfAplays a role in the growth and development of the fungus. Deletion or overexpression ofmtfAleads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased whenmtfAwas overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genesgliZandgliPwith respect to the wild type. In addition, our study showed thatmtfAis also necessary for normal protease activity inA. fumigatus; deletion ofmtfAresulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence ofmtfAcaused a decrease in virulence in theGalleria mellonellainfection model, indicating thatmtfAis necessary forA. fumigatuswild-type pathogenesis.

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