scholarly journals SpyA, a C3-Like ADP-Ribosyltransferase, Contributes to Virulence in a Mouse Subcutaneous Model of Streptococcus pyogenes Infection

2011 ◽  
Vol 79 (6) ◽  
pp. 2404-2411 ◽  
Author(s):  
Jessica S. Hoff ◽  
Mark DeWald ◽  
Steve L. Moseley ◽  
Carleen M. Collins ◽  
Jovanka M. Voyich
Keyword(s):  
Streptococcus Pyogenes ◽  
Lesion Size ◽  
Infected Tissue ◽  
Infection Model ◽  
Mrna Levels ◽  
Wild Type ◽  
Gene Encoding ◽  
Content Type ◽  
Mouse Infection Model ◽  
Adp Ribosyltransferase

ABSTRACTStreptococcus pyogenesis an important human pathogen with an expansive repertoire of verified and putative virulence factors. Here we demonstrate that a mutant deficient in the production of the streptococcal ADP-ribosyltransferase SpyA generates lesions of reduced size in a subcutaneous mouse infection model. At early stages of infection, when the difference in lesion size is first established, inflamed tissue isolated from lesions of mice infected withspyAmutant bacteria has higher levels of mRNA encoding the chemokines CXCL1 and CCL2 than does tissue isolated from mice infected with wild-type bacteria. In addition, at these early times, the mRNA levels for the gene encoding the intermediate filament vimentin are higher in the mutant-infected tissue. As wound resolution progresses, mRNA levels of the gene encoding matrix metallopeptidase 2 are lower in mutant-infected tissue. Furthermore, we demonstrate that thespyAmutant is internalized more efficiently than wild-type bacteria by HeLa cells. We conclude that SpyA contributes to streptococcal pathogenesis in the mouse subcutaneous infection model. Our observations suggest that the presence of SpyA delays wound healing in this model.

scholarly journals Pharmacodynamics of Isavuconazole in an Aspergillus fumigatus Mouse Infection Model

2015 ◽  
Vol 59 (5) ◽  
pp. 2855-2866 ◽  
Author(s):  
Seyedmojtaba Seyedmousavi ◽  
Roger J. M. Brüggemann ◽  
Jacques F. Meis ◽  
Willem J. G. Melchers ◽  
Paul E. Verweij ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Drug Exposure ◽  
Quantitative Relationship ◽  
Maximum Effect ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Type Isolate ◽  
Mouse Infection Model

ABSTRACTAzole resistance is an emerging problem inAspergillus fumigatuswhich translates into treatment failure. Alternative treatments with new azoles may improve therapeutic outcome in invasive aspergillosis (IA) even for strains with decreased susceptibility to current azoles. Thein vivoefficacy of 0.25, 1, 4, 16, 64, 128, 256, and 512 mg/kg of body weight/day prodrug isavuconazonium sulfate (BAL8557) (isavuconazole [ISA]-equivalent doses of 0.12, 0.48, 1.92, 7.68, 30.7, 61.4, 122.9, and 245.8 mg/kg/day, respectively) administered by oral gavage was assessed in an immunocompetent murine model of IA against four clinicalA. fumigatusisolates: a wild-type isolate (ISA MICEUCAST, 0.5 mg/liter) and three azole-resistant isolates harboring substitutions in thecyp51Agene: G54W (ISA MICEUCAST, 0.5 mg/liter), M220I (ISA MICEUCAST, 4 mg/liter), and TR34/L98H (ISA MICEUCAST, 8 mg/liter). The maximum effect (100% survival) was reached at a prodrug isavuconazonium sulfate dose of 64 mg/kg for the wild-type isolate, 128 mg/kg for the G54W mutant, and 256 mg/kg two times per day (q12) for the M220I mutant. A maximum response was not achieved with the TR34/L98H isolates with the highest dose of prodrug isavuconazonium sulfate (256 mg/kg q12). For a survival rate of 50%, the effective AUC0–24/MICEUCASTratio for ISA total drug was 24.73 (95% confidence interval, 22.50 to 27.18). The efficacy of isavuconazole depended on both the drug exposure and the isavuconazole MIC of the isolates. The quantitative relationship between exposure and effect (AUC0–24/MIC) can be used to optimize the treatment of human infections byA. fumigatus, including strains with decreased susceptibility.

scholarly journals c-di-AMP-Regulated K+ Importer KtrAB Affects Biofilm Formation, Stress Response, and SpeB Expression in Streptococcus pyogenes

2021 ◽  
Vol 89 (4) ◽  
Author(s):  
Sabrina Faozia ◽  
Tazin Fahmi ◽  
Gary C. Port ◽  
Kyu Hong Cho
Keyword(s):  
Stress Response ◽  
Streptococcus Pyogenes ◽  
Stress Responses ◽  
Biofilm Formation ◽  
Growth Conditions ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Double Deletion ◽  
A Subunit

ABSTRACT The second messenger cyclic di-AMP (c-di-AMP) controls biofilm formation, stress response, and virulence in Streptococcus pyogenes. The deletion of the c-di-AMP synthase gene, dacA, results in pleiotropic effects including reduced expression of the secreted protease SpeB. Here, we report a role for K+ transport in c-di-AMP-mediated SpeB expression. The deletion of ktrB in the ΔdacA mutant restores SpeB expression. KtrB is a subunit of the K+ transport system KtrAB that forms a putative high-affinity K+ importer. KtrB forms a membrane K+ channel, and KtrA acts as a cytosolic gating protein that controls the transport capacity of the system by binding ligands including c-di-AMP. SpeB induction in the ΔdacA mutant by K+ specific ionophore treatment also supports the importance of cellular K+ balance in SpeB production. The ΔdacA ΔktrB double deletion mutant not only produces wild-type levels of SpeB but also partially or fully reverts the defective ΔdacA phenotypes of biofilm formation and stress responses, suggesting that many ΔdacA phenotypes are due to cellular K+ imbalance. However, the null pathogenicity of the ΔdacA mutant in a murine subcutaneous infection model is not restored by ktrB deletion, suggesting that c-di-AMP controls not only cellular K+ balance but also other metabolic and/or virulence pathways. The deletion of other putative K+ importer genes, kup and kimA, does not phenocopy the deletion of ktrB regarding SpeB induction in the ΔdacA mutant, suggesting that KtrAB is the primary K+ importer that is responsible for controlling cellular K+ levels under laboratory growth conditions.

scholarly journals VeA Regulates Conidiation, Gliotoxin Production, and Protease Activity in the Opportunistic Human Pathogen Aspergillus fumigatus

2012 ◽  
Vol 11 (12) ◽  
pp. 1531-1543 ◽  
Author(s):  
Sourabh Dhingra ◽  
David Andes ◽  
Ana M. Calvo
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Regulatory Gene ◽  
Hydrolytic Activity ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Cellular Processes ◽  
Mouse Infection Model ◽  
Opportunistic Human Pathogen

ABSTRACTInvasive aspergillosis byAspergillus fumigatusis a leading cause of infection-related mortality in immunocompromised patients. In this study, we show thatveA, a major conserved regulatory gene that is unique to fungi, is necessary for normal morphogenesis in this medically relevant fungus. Although deletion ofveAresults in a strain with reduced conidiation, overexpression of this gene further reduced conidial production, indicating thatveAhas a major role as a regulator of development inA. fumigatusand that normal conidiation is only sustained in the presence of wild-type VeA levels. Furthermore, our studies revealed thatveAis a positive regulator in the production of gliotoxin, a secondary metabolite known to be a virulent factor inA. fumigatus. Deletion ofveAresulted in a reduction of gliotoxin production with respect to that of the wild-type control. This reduction in toxin coincided with a decrease ingliZandgliPexpression, which is necessary for gliotoxin biosynthesis. Interestingly,veAalso influences protease activity in this organism. Specifically, deletion ofveAresulted in a reduction of protease activity; this is the first report of aveAhomolog with a role in controlling fungal hydrolytic activity. AlthoughveAaffects several cellular processes inA. fumigatus, pathogenicity studies in a neutropenic mouse infection model indicated thatveAis dispensable for virulence.

scholarly journals Light-Dependent Functions of the Fusarium fujikuroi CryD DASH Cryptochrome in Development and Secondary Metabolism

2013 ◽  
Vol 79 (8) ◽  
pp. 2777-2788 ◽  
Author(s):  
Marta Castrillo ◽  
Jorge García-Martínez ◽  
Javier Avalos
Keyword(s):  
Secondary Metabolism ◽  
Nitrogen Starvation ◽  
Phytopathogenic Fungus ◽  
Fusarium Fujikuroi ◽  
Mrna Levels ◽  
Wild Type ◽  
Gene Encoding ◽  
Content Type ◽  
Gibberellin Production ◽  
In The Wild

ABSTRACTDASH (Drosophila,Arabidopsis,Synechocystis, human) cryptochromes (cry-DASHs) constitute a subgroup of the photolyase cryptochrome family with diverse light-sensing roles, found in most taxonomical groups. The genome ofFusarium fujikuroi, a phytopathogenic fungus with a rich secondary metabolism, contains a gene encoding a putative cry-DASH, named CryD. The expression of thecryDgene is induced by light in the wild type, but not in mutants of the “white collar” genewcoA. Targeted ΔcryDmutants show light-dependent phenotypic alterations, including changes in morphology and pigmentation, which disappear upon reintroduction of a wild-typecryDallele. In addition to microconidia, the colonies of the ΔcryDmutants produced under illumination and nitrogen starvation large septated spores called macroconidia, absent in wild-type colonies. The ΔcryDmutants accumulated similar amounts of carotenoids to the control strain under constant illumination, but produced much larger amounts of bikaverin under nitrogen starvation, indicating a repressing role for CryD in this biosynthetic pathway. Additionally, a moderate photoinduction of gibberellin production was exhibited by the wild type but not by the ΔcryDmutants. The phenotypic alterations of the ΔcryDmutants were only noticeable in the light, as expected from the low expression ofcryDin the dark, but did not correlate with mRNA levels for structural genes of the bikaverin or gibberellin biosynthetic pathways, suggesting the participation of CryD in posttranscriptional regulatory mechanisms. This is the first report on the participation of a cry-DASH protein in the regulation of fungal secondary metabolism.

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.

scholarly journals Modulating Pathogenesis with Mobile-CRISPRi

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Jiuxin Qu ◽  
Neha K. Prasad ◽  
Michelle A. Yu ◽  
Shuyan Chen ◽  
Amy Lyden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Effector Proteins ◽  
Infection Model ◽  
Secretion Systems ◽  
Bacterial Genomes ◽  
Gene Encoding ◽  
Content Type ◽  
Animal Infection

ABSTRACT Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models. IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

scholarly journals Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Tara L. Grinnage-Pulley ◽  
Daniel E. K. Kabotso ◽  
Chelsea L. Rintelmann ◽  
Rajarshi Roychoudhury ◽  
Robert G. Schaut ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Parasite Load ◽  
Mannose Receptor ◽  
Lesion Size ◽  
Wild Type ◽  
Cutaneous Lesions ◽  
Content Type ◽  
Latex Beads

ABSTRACTLeishmanialipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course ofLeishmaniainfection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated withLeishmania majorand trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated withL. majoralone within the first 48 h of infection. However, asL. majorinfection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected withL. majoralone, coinoculated with carrier beads andL. major, or coinoculated with trimannose-coated beads andL. major. Trimannose treatment ofL. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR−/−) mice lack the ability to detect trimannose. When MR−/−mice were infected withL. majorand treated with trimannose beads, they did not have decreased lesion size.Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.

scholarly journals Chemotherapy-Associated Changes of Histopathological Features of Mycobacterium ulcerans Lesions in a Buruli Ulcer Mouse Model

2011 ◽  
Vol 56 (2) ◽  
pp. 687-696 ◽  
Author(s):  
Marie-Thérèse Ruf ◽  
Daniela Schütte ◽  
Aurélie Chauffour ◽  
Vincent Jarlier ◽  
Baohong Ji ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
Infection Model ◽  
Histopathological Study ◽  
Content Type ◽  
Histopathological Features ◽  
Treatment Regimens ◽  
Mouse Infection Model ◽  
New Treatment

ABSTRACTCombination chemotherapy with rifampin and streptomycin (RIF-STR) for 8 weeks is currently recommended by the WHO as the first-line treatment forMycobacterium ulceransinfection (Buruli ulcer). To gain better insight into the mode of action of these antibiotics against establishedM. ulceransinfection foci and to characterize recovery of local immune responses during chemotherapy, we conducted a detailed histopathological study ofM. ulcerans-infected and RIF-STR-treated mice. Mice were inoculated withM. ulceransin the footpad and 11 weeks later treated with RIF-STR. Development of lesions during the first 11 weeks after infection and subsequent differences in disease progression between RIF-STR-treated and untreated mice were studied. Changes in histopathological features, footpad swelling, and number of CFU were analyzed. After inoculation withM. ulcerans, massive infiltrates dominated by polymorphonuclear leukocytes developed at the inoculation site but did not prevent bacterial multiplication. Huge clusters of extracellular bacteria located in large necrotic areas and surrounded by dead leukocytes developed in the untreated mice. Chemotherapy with RIF-STR led to a rapid drop in CFU associated with loss of solid Ziehl-Neelsen staining of acid-fast bacilli. Development of B-lymphocyte clusters and of macrophage accumulations surrounding the mycobacteria demonstrated the resolution of local immune suppression. Results demonstrate that the experimentalM. ulceransmouse infection model will be a valuable tool to investigate efficacy of new treatment regimens and of candidate vaccines.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals Impact of Glutamine Transporters on Pneumococcal Fitness under Infection-Related Conditions

2010 ◽  
Vol 79 (1) ◽  
pp. 44-58 ◽  
Author(s):  
Tobias Härtel ◽  
Matthias Klein ◽  
Uwe Koedel ◽  
Manfred Rohde ◽  
Lothar Petruschka ◽  
...  
Keyword(s):  
White Blood Cells ◽  
Survival Rates ◽  
Genomic Analysis ◽  
Infection Model ◽  
Wild Type ◽  
Survival Times ◽  
Reverse Transcription Pcr ◽  
Mouse Infection Model ◽  
The Impact ◽  
Glutamine Uptake

ABSTRACTThe genomic analysis ofStreptococcuspneumoniaepredicted six putative glutamine uptake systems, which are expressed underinvitroconditions, as shown here by reverse transcription-PCR. Four of these operons consist ofglnHPQ, while two lackglnH, which encodes a soluble glutamine-binding protein. Here, we studied the impact of two of these glutamine ATP-binding cassette transporters onS.pneumoniaeD39 virulence and phagocytosis, which consist of GlnQ and a translationally fused protein of GlnH and GlnP. Mice infected intranasally with D39Δgln0411/0412showed significantly increased survival times and a significant delay in the development of pneumococcal pneumonia compared to those infected with D39, as observed in real time using bioluminescent pneumococci. In a mouse sepsis model, the mutant D39Δgln0411/0412showed only moderate but significant attenuation. In contrast, the D39Δgln1098/1099knockout strain was massively attenuated in the pneumonia and septicemia mouse infection model. To cause pneumonia or sepsis with D39Δgln1098/1099, infection doses 100- to 10,000-fold higher than those used for wild-type strain D39 were required. In an experimental mouse meningitis model, D39Δgln1098/1099produced decreased levels of white blood cells in cerebrospinal fluid and showed decreased numbers of bacteria in the bloodstream compared to D39 and D39Δgln0411/0412. Phagocytosis experiments revealed significantly decreased intracellular survival rates of mutants D39Δgln1098/1099and D39Δgln0411/0412compared to wild-type D39, suggesting that the deficiency of Gln uptake systems impairs resistance to oxidative stress. Taken together, our results demonstrate that both glutamine uptake systems are required for full virulence of pneumococci but exhibit different impacts on the pathogenesis of pneumococci underinvivoconditions.

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