scholarly journals The replication of the chromosome of Pseudomonas aeruginosa strain 1: II. Sequential mutagenesis of synchronized cultures

1975 ◽  
Vol 25 (3) ◽  
pp. 215-227 ◽  
Author(s):  
R. J. Booker ◽  
J. S. Loutit
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Chromosome ◽  
Linkage Groups ◽  
Gene Frequencies ◽  
Replication Point ◽  
Sequential Mutagenesis ◽  
Exponential Populations ◽  
Synchronized Cultures ◽  
Increased Susceptibility

SUMMARYThe order of replication of a series of genes in Pseudomonas aeruginosa has been studied in synchronized cultures using a method based on the technique of sequential mutagenesis. This technique relies on the increased susceptibility of the replication point of the bacterial chromosome to mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine. The genes studied were those previously mapped by conjugation and whose order of replication had been studied by an investigation of gene frequencies in exponential populations. The results are consistent with the idea that there is two-way replication of the chromosome of P. aeruginosa starting at a point near trp-1 and arg-6. They also confirm that the two linkage groups which have been found by conjugation replicate at different times. If the assumption is made that there is only one chromosome in P. aeruginosa, the results can be used to show how the two linkage groups may possibly be joined together and the order is such that there would have to be two sites of attachment for the sex factor FP2.

scholarly journals Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium

2009 ◽  
Vol 78 (3) ◽  
pp. 939-953 ◽  
Author(s):  
Iwona Bucior ◽  
Keith Mostov ◽  
Joanne N. Engel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mdck Cells ◽  
Three Dimensional ◽  
Opportunistic Pathogen ◽  
Necessary And Sufficient ◽  
Plastic Surfaces ◽  
Pharmacologic Inhibition ◽  
Increased Susceptibility ◽  
Polarized Epithelium

ABSTRACT Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.

scholarly journals Fosfomycin and Tobramycin in Combination Downregulate Nitrate Reductase GenesnarGandnarH, Resulting in Increased Activity against Pseudomonas aeruginosa under Anaerobic Conditions

2013 ◽  
Vol 57 (11) ◽  
pp. 5406-5414 ◽  
Author(s):  
Gerard McCaughey ◽  
Deirdre F. Gilpin ◽  
Thamarai Schneiders ◽  
Lucas R. Hoffman ◽  
Matt McKevitt ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrate Reductase ◽  
Growth Curves ◽  
Anaerobic Growth ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Transposon Mutants ◽  
Increased Activity ◽  
Increased Susceptibility

ABSTRACTThe activity of aminoglycosides, which are used to treatPseudomonas aeruginosarespiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lungin vivo. In contrast, a 4:1 (wt/wt) combination of fosfomycin and tobramycin (F:T), which is under investigation for use in the treatment of CF lung infection, has increased activity againstP. aeruginosaunder anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests, including nitrate utilization assays, growth curves, and susceptibility testing. Notably, growth in subinhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (P< 0.05) nitrate reductase genesnarGandnarH, which are essential for normal anaerobic growth ofP. aeruginosa. Under anaerobic conditions, F:T significantly decreased (P< 0.001) nitrate utilization inP. aeruginosastrains PAO1, PA14, and PA14lasR::Gm, a mutant known to exhibit increased nitrate utilization. A similar effect was observed with two clinicalP. aeruginosaisolates. Growth curves indicate that nitrate reductase transposon mutants had reduced growth under anaerobic conditions, with these mutants also having increased susceptibility to F:T compared to the wild type under similar conditions. The results of this study suggest that downregulation of nitrate reductase genes resulting in reduced nitrate utilization is the mechanism underlying the increased activity of F:T under anaerobic conditions.

Increased susceptibility to Pseudomonas aeruginosa infection under hindlimb-unloading conditions

2003 ◽  
Vol 95 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Hernan Aviles ◽  
Tesfaye Belay ◽  
Kimberly Fountain ◽  
Monique Vance ◽  
Gerald Sonnenfeld
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune System ◽  
Critical Role ◽  
Hindlimb Unloading ◽  
Washington Dc ◽  
Time To Death ◽  
Allergy Clin ◽  
Pseudomonas Aeruginosa Infection ◽  
And Control ◽  
Increased Susceptibility

It has been reported that spaceflight conditions alter the immune system and resistance to infection [Belay T, Aviles H, Vance M, Fountain K, and Sonnenfeld G. J Allergy Clin Immunol 170: 262–268, 2002; Hankins WR and Ziegelschmid JF. In: Biomedical Results of Apollo. Washington, DC: NASA, 1975, p. 43–81 . (NASA Spec. Rep. SP-368)]. Ground-based models, including the hindlimb-unloading model, have become important tools for increasing understanding of how spaceflight conditions can influence physiology. The objective of the present study was to determine the effect of hindlimb unloading on the susceptibility of mice to Pseudomonas aeruginosa infection. Hindlimb-unloaded and control mice were subcutaneously infected with 1 LD50 of P. aeruginosa. Survival, bacterial organ load, and antibody and corticosterone levels were compared among the groups. Hindlimb unloading had detrimental effects for infected mice. Animals in the hindlimb-unloaded group, compared with controls, 1) showed significantly increased mortality and reduced time to death, 2) had increased levels of corticosterone, and 3) were much less able to clear bacteria from the organs. These results suggest that hindlimb unloading may induce the production of corticosterone, which may play a critical role in the modulation of the immune system leading to increased susceptibility to P. aeruginosa infection.

scholarly journals Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in Pseudomonas aeruginosa

2000 ◽  
Vol 44 (9) ◽  
pp. 2242-2246 ◽  
Author(s):  
Nobuhisa Masuda ◽  
Eiko Sakagawa ◽  
Satoshi Ohya ◽  
Naomasa Gotoh ◽  
Hideto Tsujimoto ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Rabbit Serum ◽  
Deficient Mutant ◽  
Efflux System ◽  
Intrinsic Resistance ◽  
Strain Pao1 ◽  
Isogenic Mutants ◽  
Increased Susceptibility

ABSTRACT To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/ormexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of themexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of amexAB-oprM- and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

scholarly journals Determinants of Intrinsic Aminoglycoside Resistance in Pseudomonas aeruginosa

2012 ◽  
Vol 56 (11) ◽  
pp. 5591-5602 ◽  
Author(s):  
Thomas Krahn ◽  
Christie Gilmour ◽  
Justin Tilak ◽  
Sebastien Fraud ◽  
Nicholas Kerr ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Envelope ◽  
Acquired Resistance ◽  
Insertion Mutant ◽  
Deletion Mutants ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Single Deletion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACTScreening of a transposon insertion mutant library ofPseudomonas aeruginosafor increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism (lptA,faoA), phosphate uptake (pstB), and two-component regulators (amgRS, PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants (pstB,faoA, PA0392,amgR) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome inP. aeruginosa. Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.

scholarly journals Polymyxin Susceptibility in Pseudomonas aeruginosa Linked to the MexXY-OprM Multidrug Efflux System

2015 ◽  
Vol 59 (12) ◽  
pp. 7276-7289 ◽  
Author(s):  
Keith Poole ◽  
Calvin Ho-Fung Lau ◽  
Christie Gilmour ◽  
Youai Hao ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Mechanism ◽  
Polymyxin B ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Content Type ◽  
Polymyxin E ◽  
Increased Susceptibility ◽  
Multidrug Efflux System

ABSTRACTThe ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced themexXYgenes of the MexXY-OprM multidrug efflux system inPseudomonas aeruginosaand increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci,arnBCADTEFand PA4773-74. Consistent with the SPC-promoted reduction inarnand PA4773-74 expression being linked tomexXY, expression of these LPS modification loci was moderated in a mutant constitutively expressingmexXYand enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lackingarnBand/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lackingmexXYwas unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance ‘mechanism’ is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-typeP. aeruginosabut not in the ΔmexXYmutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-inducedmexXYexpression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility.

scholarly journals Vitamin D Deficiency Does Not Result in a Breach of Host Defense in Murine Models of Pneumonia

2016 ◽  
Vol 84 (11) ◽  
pp. 3097-3104 ◽  
Author(s):  
Julia Niederstrasser ◽  
Christian Herr ◽  
Lisa Wolf ◽  
Claus M. Lehr ◽  
Christoph Beisswenger ◽  
...  
Keyword(s):  
Vitamin D ◽  
Pseudomonas Aeruginosa ◽  
Streptococcus Pneumoniae ◽  
Host Defense ◽  
Murine Models ◽  
Deficient Diet ◽  
Content Type ◽  
Cell Counts ◽  
The Impact ◽  
Increased Susceptibility

Vitamin D (VitD) has a role in the regulation of calcium and phosphate metabolism and in addition impacts the activity of the immune system. VitD deficiency might be linked to increased susceptibility to respiratory tract infection. The aim of the present study was to characterize the impact of VitD deficiency on the susceptibility to bacterial infection in murine models. C57BL/6N mice were fed a diet with or without VitD for 10 weeks. The VitD-deficient or -sufficient mice were infected with Pseudomonas aeruginosa or Streptococcus pneumoniae . The colonization and inflammatory response in the lung were analyzed at defined time points. The serum 25-hydroxy-VitD concentration was significantly lower in mice on the VitD-deficient diet. In infection experiments with Pseudomonas aeruginosa or Streptococcus pneumoniae , no differences could be observed in the numbers of viable bacteria or in differential cell counts in the bronchoalveolar lavage fluids. Measurements of inflammatory cytokines (KC and interleukin-1β [IL-1β]) did not show significant differences between the groups. In conclusion, VitD-deficient animals did not show significantly increased susceptibility to infection or an altered course of infection. The immune systems of humans and mice likely respond differently to VitD. Murine models are likely not appropriate for drawing conclusions on the role of VitD in human pulmonary host defense.

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