scholarly journals Benefit of Having Multiple ampD Genes for Acquiring β-Lactam Resistance without Losing Fitness and Virulence in Pseudomonas aeruginosa

2008 ◽  
Vol 52 (10) ◽  
pp. 3694-3700 ◽  
Author(s):  
Bartolomé Moya ◽  
Carlos Juan ◽  
Sebastián Albertí ◽  
José L. Pérez ◽  
Antonio Oliver
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mouse Model ◽  
Resistance Mechanism ◽  
Systemic Infection ◽  
Triple Mutant ◽  
Biological Cost ◽  
Sequential Inactivation ◽  
High Level ◽  
Insight Into

ABSTRACT The inactivation of ampD in Pseudomonas aeruginosa leads to a partially derepressed phenotype, characterized by a moderately high level basal ampC expression that is still further inducible, due to the presence of two additional ampD genes in this species (ampDh2 and ampDh3). The sequential inactivation of the three ampD genes was shown to lead to a stepwise upregulation of ampC expression, reaching full derepression in the triple mutant. To gain insight into the biological role of P. aeruginosa AmpD multiplicity, we determined the effects of the inactivation of the ampD genes on fitness and virulence. We show that, in contrast to what was previously documented for Salmonella spp., the inactivation of ampD in P. aeruginosa does not affect fitness or virulence in a mouse model of systemic infection. This lack of effect was demonstrated to be dependent on the presence of the additional ampD genes (ampDh2 and ampDh3), since the double and the triple ampD mutants completely lost their biological competitiveness and virulence; full ampC derepression and disruption of the AmpD peptidoglycan recycling system itself are both found to cause a major biological cost. Furthermore, among the ampD genes, ampDh3 is found to be the most relevant for virulence in P. aeruginosa. Therefore, as a consequence of the presence of additional ampD genes, partial ampC derepression mediated by ampD inactivation confers a biologically efficient resistance mechanism on P. aeruginosa.

scholarly journals Dissemination of Rat Cytomegalovirus through Infected Granulocytes and Monocytes In Vitro and In Vivo

2003 ◽  
Vol 77 (20) ◽  
pp. 11274-11278 ◽  
Author(s):  
B. W. A. van der Strate ◽  
J. L. Hillebrands ◽  
S. S. Lycklama à Nijeholt ◽  
L. Beljaars ◽  
C. A. Bruggeman ◽  
...  
Keyword(s):  
Intravenous Injection ◽  
Systemic Infection ◽  
Insight Into

ABSTRACT The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.

scholarly journals Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

2003 ◽  
Vol 71 (5) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sachiko Miyata ◽  
Monika Casey ◽  
Dara W. Frank ◽  
Frederick M. Ausubel ◽  
Eliana Drenkard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Galleria Mellonella ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Triple Mutant ◽  
Type Iii ◽  
Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

scholarly journals Genomic and Molecular Perspectives of Host-pathogen Interaction and Resistance Strategies against White Rust in Oilseed Mustard

2020 ◽  
Vol 21 (3) ◽  
pp. 179-193
Author(s):  
Chatterjee Anupriya ◽  
Nirwan Shradha ◽  
Bandyopadhyay Prasun ◽  
Agnihotri Abha ◽  
Sharma Pankaj ◽  
...  
Keyword(s):  
Crop Protection ◽  
Resistance Mechanism ◽  
Pathogen Resistance ◽  
Susceptibility Genes ◽  
Rust Disease ◽  
Disease Response ◽  
White Rust ◽  
Oilseed Brassicas ◽  
Insight Into

: Oilseed brassicas stand as the second most valuable source of vegetable oil and the third most traded one across the globe. However, the yield can be severely affected by infections caused by phytopathogens. White rust is a major oomycete disease of oilseed brassicas resulting in up to 60% yield loss globally. So far, success in the development of oomycete resistant Brassicas through conventional breeding has been limited. Hence, there is an imperative need to blend conventional and frontier biotechnological means to breed for improved crop protection and yield. : This review provides a deep insight into the white rust disease and explains the oomycete-plant molecular events with special reference to Albugo candida describing the role of effector molecules, A. candida secretome, and disease response mechanism along with nucleotide-binding leucine-rich repeat receptor (NLR) signaling. Based on these facts, we further discussed the recent progress and future scopes of genomic approaches to transfer white rust resistance in the susceptible varieties of oilseed brassicas, while elucidating the role of resistance and susceptibility genes. Novel genomic technologies have been widely used in crop sustainability by deploying resistance in the host. Enrichment of NLR repertoire, over-expression of R genes, silencing of avirulent and disease susceptibility genes through RNA interference and CRSPR-Cas are technologies which have been successfully applied against pathogen-resistance mechanism. The article provides new insight into Albugo and Brassica genomics which could be useful for producing high yielding and WR resistant oilseed cultivars across the globe.

scholarly journals Stepwise Upregulation of the Pseudomonas aeruginosa Chromosomal Cephalosporinase Conferring High-Level β-Lactam Resistance Involves Three AmpD Homologues

2006 ◽  
Vol 50 (5) ◽  
pp. 1780-1787 ◽  
Author(s):  
Carlos Juan ◽  
Bartolomé Moyá ◽  
José L. Pérez ◽  
Antonio Oliver
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Mechanisms ◽  
Natural Resistance ◽  
Wild Type ◽  
Triple Mutant ◽  
Major Threat ◽  
Development Of Resistance ◽  
One Step ◽  
High Level ◽  
Very High

ABSTRACT Development of resistance to the antipseudomonal penicillins and cephalosporins mediated by hyperproduction of the chromosomal cephalosporinase AmpC is a major threat to the successful treatment of Pseudomonas aeruginosa infections. Although ampD inactivation has been previously found to lead to a partially derepressed phenotype characterized by increased AmpC production but retaining further inducibility, the regulation of ampC in P. aeruginosa is far from well understood. We demonstrate that ampC expression is coordinately repressed by three AmpD homologues, including the previously described protein AmpD plus two additional proteins, designated AmpDh2 and AmpDh3. The three AmpD homologues are responsible for a stepwise ampC upregulation mechanism ultimately leading to constitutive hyperexpression of the chromosomal cephalosporinase and high-level antipseudomonal β-lactam resistance, as shown by analysis of the three single ampD mutants, the three double ampD mutants, and the triple ampD mutant. This is achieved by a three-step escalating mechanism rendering four relevant expression states: basal-level inducible expression (wild type), moderate-level hyperinducible expression with increased antipseudomonal β-lactam resistance (ampD mutant), high-level hyperinducible expression with high-level β-lactam resistance (ampD ampDh3 double mutant), and very high-level (more than 1,000-fold compared to the wild type) derepressed expression (triple mutant). Although one-step inducible-derepressed expression models are frequent in natural resistance mechanisms, this is the first characterized example in which expression of a resistance gene can be sequentially amplified through multiple steps of derepression.

scholarly journals Cytoplasmic Copper Detoxification in Salmonella Can Contribute to SodC Metalation but Is Dispensable during Systemic Infection

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Luke A. Fenlon ◽  
James M. Slauch
Keyword(s):  
Superoxide Dismutase ◽  
Immune Cells ◽  
Gastrointestinal Disease ◽  
Systemic Infection ◽  
Copper Binding ◽  
Wild Type ◽  
Triple Mutant ◽  
Content Type

ABSTRACT Salmonella enterica serovar Typhimurium is a leading cause of foodborne disease worldwide. Severe infections result from the ability of S. Typhimurium to survive within host immune cells, despite being exposed to various host antimicrobial factors. SodCI, a copper-zinc-cofactored superoxide dismutase, is required to defend against phagocytic superoxide. SodCII, an additional periplasmic superoxide dismutase, although produced during infection, does not function in the host. Previous studies suggested that CueP, a periplasmic copper binding protein, facilitates acquisition of copper by SodCII. CopA and GolT, both inner membrane ATPases that pump copper from the cytoplasm to the periplasm, are a source of copper for CueP. Using in vitro SOD assays, we found that SodCI can also utilize CueP to acquire copper. However, both SodCI and SodCII have a significant fraction of activity independent of CueP and cytoplasmic copper export. We utilized a series of mouse competition assays to address the in vivo role of CueP-mediated SodC activation. A copA golT cueP triple mutant was equally as competitive as the wild type, suggesting that sufficient SodCI is active to defend against phagocytic superoxide independent of CueP and cytoplasmic copper export. We also confirmed that a strain containing a modified SodCII, which is capable of complementing a sodCI deletion, was fully virulent in a copA golT cueP background competed against the wild type. These competitions also address the potential impact of cytoplasmic copper toxicity within the phagosome. Our data suggest that Salmonella does not encounter inhibitory concentrations of copper during systemic infection. IMPORTANCE Salmonella is a leading cause of gastrointestinal disease worldwide. In severe cases, Salmonella can cause life-threatening systemic infections, particularly in very young children, the elderly, or people who are immunocompromised. To cause disease, Salmonella must survive the hostile environment inside host immune cells, a location in which most bacteria are killed. Our work examines how one particular metal, copper, is acquired by Salmonella to activate a protein important for survival within immune cells. At high levels, copper itself can inhibit Salmonella. Using a strain of Salmonella that cannot detoxify intracellular copper, we also addressed the in vivo role of copper as an antimicrobial agent.

scholarly journals PmrB Mutations Promote Polymyxin Resistance of Pseudomonas aeruginosa Isolated from Colistin-Treated Cystic Fibrosis Patients

2011 ◽  
Vol 56 (2) ◽  
pp. 1019-1030 ◽  
Author(s):  
Samuel M. Moskowitz ◽  
Mark K. Brannon ◽  
Nandini Dasgupta ◽  
Miyuki Pier ◽  
Nicole Sgambati ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Clinical Isolates ◽  
Gain Of Function ◽  
Content Type ◽  
Regulatory Systems ◽  
Repeated Passage ◽  
High Level

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance,P. aeruginosaisolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-functionpmrBalleles that conferred polymyxin resistance to strains with a wild-type orpmrABdeletion background. Double mutantpmrBalleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutantpmrBalleles induced transcription from the promoter of thearnBoperon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate thatpmrBgain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains ofP. aeruginosa.

scholarly journals GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues

2012 ◽  
Vol 57 (1) ◽  
pp. 396-401 ◽  
Author(s):  
Carine Bebrone ◽  
Pierre Bogaerts ◽  
Heinrich Delbrück ◽  
Sandra Bennink ◽  
Michaël B. Kupper ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lower Respiratory Tract ◽  
Resistance Mechanism ◽  
Single Amino Acid ◽  
Content Type ◽  
Reference Center ◽  
National Reference ◽  
National Reference Center ◽  
Similar Kinetic

ABSTRACTA clinical isolate ofPseudomonas aeruginosarecovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its resistance mechanism. PCR sequencing identified a new GES variant, GES-18, which differs from the carbapenem-hydrolyzing enzyme GES-5 by a single amino acid substitution (Val80Ile, in the numbering according to Ambler) and from GES-1 by two substitutions (Val80Ile and Gly170Ser). Detailed kinetic characterization showed that GES-18 and GES-5 hydrolyze imipenem and cefoxitin with similar kinetic parameters and that GES-18 was less susceptible than GES-1 to classical β-lactamase inhibitors such as clavulanate and tazobactam. The overall structure of GES-18 is similar to the solved structures of GES-1 and GES-2, the Val80Ile and Gly170Ser substitutions causing only subtle local rearrangements. Notably, the hydrolytic water molecule and the Glu166 residue were slightly displaced compared to their counterparts in GES-1. Our kinetic and crystallographic data for GES-18 highlight the pivotal role of the Gly170Ser substitution which distinguishes GES-5 and GES-18 from GES-1.

Limited contribution of the outer-membrane penetration barrier towards intrinsic antibiotic resistance in Pseudomonas aeruginosa

1982 ◽  
Vol 28 (2) ◽  
pp. 169-175 ◽  
Author(s):  
R. Allan Scudamore ◽  
Morris Goldner
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Growth Curves ◽  
Beta Lactam ◽  
Intrinsic Resistance ◽  
Beta Lactam Antibiotics ◽  
High Level

The role of the outer membrane (OM) was investigated in relation to the high level of intrinsic antibiotic resistance of Pseudomonas aeruginosa ATCC 9027. OM penetration barriers were measured by comparing turbidimetric growth curves of EDTA-treated and normal cells exposed to carbenicillin, moxalactam (LY 127935), gentamicin, tobramycin, rifampin, novobiocin, and vancomycin. OM barriers were also measured for carbenicillin and moxalactam in P. aeruginosa strain K 799/61, a hypersusceptible mutant presumed to have lost its penetration barrier in the cell envelope. Most antibiotics penetrated the OM efficiently and there was little difference between the two strains. The evidence therefore suggests that intrinsic resistance of P. aeruginosa, especially to the beta-lactam antibiotics, is not mainly due to the OM. A penetration barrier situated deeper within the cell envelope is hypothesized, the size of which in relation to any antibiotic may be estimated by comparing the IC50 values of EDTA-treated cells of the two strains.

scholarly journals A novel mouse model of conditional IRAK-M deficiency in myeloid cells: application in lung Pseudomonas aeruginosa infection

2016 ◽  
Vol 23 (2) ◽  
pp. 206-215 ◽  
Author(s):  
Di Jiang ◽  
Jennifer Matsuda ◽  
Reena Berman ◽  
Niccolette Schaefer ◽  
Connor Stevenson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mouse Model ◽  
Bacterial Infection ◽  
Knockout Mice ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Conditional Knockout ◽  
Negative Regulator ◽  
Innate Defense

Myeloid cells such as macrophages are critical to innate defense against infection. IL-1 receptor-associated kinase M (IRAK-M) is a negative regulator of TLR signaling during bacterial infection, but the role of myeloid cell IRAK-M in bacterial infection is unclear. Our goal was to generate a novel conditional knockout mouse model to define the role of myeloid cell IRAK-M during bacterial infection. Myeloid cell-specific IRAK-M knockout mice were generated by crossing IRAK-M floxed mice with LysM–Cre knock-in mice. The resulting LysM–Cre+/IRAK-Mfl/wt and control (LysM–Cre–/IRAK-Mfl/wt) mice were intranasally infected with Pseudomonas aeruginosa (PA). IRAK-M deletion, inflammation, myeloperoxidase (MPO) activity and PA load were measured in leukocytes, bronchoalveolar lavage (BAL) fluid and lungs. PA killing assay with BAL fluid was performed to determine mechanisms of IRAK-M-mediated host defense. IRAK-M mRNA and protein levels in alveolar and lung macrophages were significantly reduced in LysM–Cre+/IRAK-Mfl/wt mice compared with control mice. Following PA infection, LysM–Cre+/IRAK-Mfl/wt mice have enhanced lung neutrophilic inflammation, including MPO activity, but reduced PA load. The increased lung MPO activity in LysM–Cre+/IRAK-Mfl/wt mouse BAL fluid reduced PA load. Generation of IRAK-M conditional knockout mice will enable investigators to determine precisely the function of IRAK-M in myeloid cells and other types of cells during infection and inflammation.

Extrasynaptic CaMKIIα is involved in the antidepressant effects of ketamine by downregulating GluN2B receptors in an LPS-induced depression model

2020 ◽  
Author(s):  
Xiao-Hui Tang ◽  
Guang-Fen Zhang ◽  
Ning Xu ◽  
Gui-Fang Duan ◽  
Min Jia ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Mouse Model ◽  
Cellular Mechanism ◽  
Antidepressant Effect ◽  
Immunoprecipitation Assay ◽  
Antidepressant Effects ◽  
High Level ◽  
Induced Changes ◽  
The Relationship

Abstract Background A subanesthetic dose of ketamine provides rapid and effective antidepressant effects, but the molecular mechanism of this treatment remains elusive. Methods In this study, we investigated the role of CaMKIIα in the antidepressant effects of ketamine using an LPS-induced mouse model of depression, explored the different changes of CaMKIIα in the synaptic and extrasynaptic regions of the hippocampus, and clarified the relationship between CaMKIIα and GluN2B from extrasynaptic perspective. Results Ketamine (10 mg/kg, i.p.) administration attenuated the LPS-induced increase in extrasynaptic CaMKIIα activity (p-CaMKIIα) and extrasynaptic GluN2B localization and phosphorylation and that ketamine exerted antidepressant effects. Immunoprecipitation assay revealed that in the extrasynaptic region of the hippocampus, p-CaMKIIα bound to GluN2B, and ketamine administration attenuated the enhanced interaction between p-CaMKIIα and GluN2B induced by LPS. KN93, a CaMKIIα inhibitor, could also reverse the high level of extrasynaptic p-CaMKIIα, reduce hippocampal extrasynaptic GluN2B localization and phosphorylation, and exert antidepressant effects. Additional changes downstream of the ketamine-induced changes in extrasynaptic GluN2B included rescuing the downregulated expression of p-CREB, BDNF, and GluR1 and reversing the impaired induction of LTP in the hippocampus induced by LPS. Conclusion These results indicate that extrasynaptic CaMKIIα plays a key role in the cellular mechanism of ketamine's antidepressant effect and is related to the down-regulation of extrasynaptic GluN2B localization and phosphorylation and further affects synaptic plasticity.

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