scholarly journals Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

2016 ◽  
Vol 84 (8) ◽  
pp. 2324-2335 ◽  
Author(s):  
Fabrizia Minandri ◽  
Francesco Imperi ◽  
Emanuela Frangipani ◽  
Carlo Bonchi ◽  
Daniela Visaggio ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mouse Model ◽  
Human Serum ◽  
Iron Uptake ◽  
Lung Infection ◽  
Null Mutant ◽  
Wild Type ◽  
Heme Uptake ◽  
Content Type

Pseudomonas aeruginosais a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, andP. aeruginosaexpresses multiple iron uptake systems, whose role in lung infection deserves further investigation.P. aeruginosaFe3+uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer.P. aeruginosaalso has the FeoB transporter for Fe2+acquisition. To assess the roles of individual iron uptake systems inP. aeruginosalung infection, single and double deletion mutants were generated inP. aeruginosaPAO1 and characterizedin vitro, using iron-poor media and human serum, andin vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and thefeoB,hasR phuR(heme uptake), andpchD(pyochelin) mutants grewin vitroand caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, apvdA fpvRdouble mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion offpvRin thepvdAbackground partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis ofP. aeruginosalung infection by combining iron transport and virulence-inducing capabilities.

scholarly journals The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Mauricio A. Navarro ◽  
Jihong Li ◽  
Juliann Beingesser ◽  
Bruce A. McClane ◽  
Francisco A. Uzal
Keyword(s):  
Quorum Sensing ◽  
Mouse Model ◽  
Clostridium Perfringens ◽  
Regulatory System ◽  
Type A ◽  
Gas Gangrene ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type

ABSTRACT Clostridium perfringens type A is involved in gas gangrene in humans and animals. Following a traumatic injury, rapid bacterial proliferation and exotoxin production result in severe myonecrosis. C. perfringens alpha toxin (CPA) and perfringolysin (PFO) are the main virulence factors responsible for the disease. Recent in vitro studies have identified an Agr-like quorum-sensing (QS) system in C. perfringens that regulates the production of both toxins. The system is composed of an AgrB membrane transporter and an AgrD peptide that interacts with a two-component regulatory system in response to fluctuations in the cell population density. In addition, a synthetic peptide named 6-R has been shown to interfere with this signaling mechanism, affecting the function of the Agr-like QS system in vitro. In the present study, C. perfringens type A strain ATCC 3624 and an isogenic agrB-null mutant were tested in a mouse model of gas gangrene. When mice were intramuscularly challenged with 106 CFU of wild-type ATCC 3624, severe myonecrosis and leukocyte aggregation occurred by 4 h. Similar numbers of an agrB-null mutant strain produced significantly less severe changes in the skeletal muscle of challenged mice. Complementation of the mutant to regain agrB expression restored virulence to wild-type levels. The burdens of all three C. perfringens strains in infected muscle were similar. In addition, animals injected intramuscularly with wild-type ATCC 3624 coincubated with the 6-R peptide developed less severe microscopic changes. This study provides the first in vivo evidence that the Agr-like QS system is important for C. perfringens type A-mediated gas gangrene. IMPORTANCE Clostridium perfringens type A strains produce toxins that are responsible for clostridial myonecrosis, also known as gas gangrene. Toxin production is regulated by an Agr-like quorum-sensing (QS) system that responds to changes in cell population density. In this study, we investigated the importance of this QS system in a mouse model of gas gangrene. Mice challenged with a C. perfringens strain with a nonfunctional regulatory system developed less severe changes in the injected skeletal muscle compared to animals receiving the wild-type strain. In addition, a synthetic peptide was able to decrease the effects of the QS in this disease model. These studies provide new understanding of the pathogenesis of gas gangrene and identified a potential therapeutic target to prevent the disease.

scholarly journals Requirement of the Pseudomonas aeruginosa CbrA Sensor Kinase for Full Virulence in a Murine Acute Lung Infection Model

2013 ◽  
Vol 82 (3) ◽  
pp. 1256-1267 ◽  
Author(s):  
Amy T. Y. Yeung ◽  
Laure Janot ◽  
Olga M. Pena ◽  
Anke Neidig ◽  
Irena Kukavica-Ibrulj ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lung Infection ◽  
Lung Model ◽  
Transcriptional Analysis ◽  
Infection Model ◽  
Sensor Kinase ◽  
Wild Type ◽  
Bacterial Killing ◽  
Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that is a major cause of respiratory tract and other nosocomial infections. The sensor kinase CbrA is a central regulator of carbon and nitrogen metabolism andin vitroalso regulates virulence-related processes inP. aeruginosa. Here, we investigated the role of CbrA in two murine models of infection. In both peritoneal infections in leukopenic mice and lung infection models, thecbrAmutant was less virulent since substantially larger numbers ofcbrAmutant bacteria were required to cause the same level of infection as wild-type or complemented bacteria. In contrast, in the chronic rat lung model thecbrAmutant grew and persisted as well as the wild type, indicating that the decrease ofin vivovirulence of thecbrAmutant did not result from growth deficiencies on particular carbon substrates observedin vitro. In addition, a mutant in the cognate response regulator CbrB showed no defect in virulence in the peritoneal infection model, ruling out the involvement of certain alterations of virulence properties in thecbrAmutant including defective swarming motility, increased biofilm formation, and cytotoxicity, since these alterations are controlled through CbrB. Further investigations indicated that the mutant was more susceptible to uptake by phagocytesin vitro, resulting in greater overall bacterial killing. Consistent with the virulence defect, it took a smaller number ofDictyostelium discoideumamoebae to kill thecbrAmutant than to kill the wild type. Transcriptional analysis of thecbrAmutant duringD. discoideuminfection led to the conclusion that CbrA played an important role in the iron metabolism, protection ofP. aeruginosaagainst oxidative stress, and the regulation of certain virulence factors.

scholarly journals Bactericidal Activity, Absence of Serum Effect, and Time-Kill Kinetics of Ceftazidime-Avibactam against β-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

2014 ◽  
Vol 58 (9) ◽  
pp. 5297-5305 ◽  
Author(s):  
Tiffany R. Keepers ◽  
Marcela Gomez ◽  
Chris Celeri ◽  
Wright W. Nichols ◽  
Kevin M. Krause
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Serum ◽  
Bactericidal Activity ◽  
Minimum Bactericidal Concentration ◽  
Wild Type ◽  
Content Type ◽  
Positive Isolate ◽  
New Treatment ◽  
Time Kill ◽  
Kinetics Of

ABSTRACTAvibactam, a non-β-lactam β-lactamase inhibitor with activity against extended-spectrum β-lactamases (ESBLs), KPC, AmpC, and some OXA enzymes, extends the antibacterial activity of ceftazidime against most ceftazidime-resistant organisms producing these enzymes. In this study, the bactericidal activity of ceftazidime-avibactam against 18Pseudomonas aeruginosaisolates and 15Enterobacteriaceaeisolates, including wild-type isolates and ESBL, KPC, and/or AmpC producers, was evaluated. Ceftazidime-avibactam MICs (0.016 to 32 μg/ml) were lower than those for ceftazidime alone (0.06 to ≥256 μg/ml) against all isolates except for 2P. aeruginosaisolates (1blaVIM-positive isolate and 1blaOXA-23-positive isolate). The minimum bactericidal concentration/MIC ratios of ceftazidime-avibactam were ≤4 for all isolates, indicating bactericidal activity. Human serum and human serum albumin had a minimal effect on ceftazidime-avibactam MICs. Ceftazidime-avibactam time-kill kinetics were evaluated at low MIC multiples and showed time-dependent reductions in the number of CFU/ml from 0 to 6 h for all strains tested. A ≥3-log10decrease in the number of CFU/ml was observed at 6 h for allEnterobacteriaceae, and a 2-log10reduction in the number of CFU/ml was observed at 6 h for 3 of the 6P. aeruginosaisolates. Regrowth was noted at 24 h for some of the isolates tested in time-kill assays. These data demonstrate the potent bactericidal activity of ceftazidime-avibactam and support the continued clinical development of ceftazidime-avibactam as a new treatment option for infections caused byEnterobacteriaceaeandP. aeruginosa, including isolates resistant to ceftazidime by mechanisms dependent on avibactam-sensitive β-lactamases.

scholarly journals Availability of Zinc Impacts Interactions between Streptococcus sanguinis and Pseudomonas aeruginosa in Coculture

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Kewei Li ◽  
Alex H. Gifford ◽  
Thomas H. Hampton ◽  
George A. O’Toole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Zinc Uptake ◽  
Microbial Interactions ◽  
Growth Defect ◽  
Zinc Toxicity ◽  
Wild Type ◽  
Content Type ◽  
Airway Infections ◽  
Zinc Levels

ABSTRACT Airway infections associated with cystic fibrosis (CF) are polymicrobial. We reported previously that clinical isolates of Pseudomonas aeruginosa promote the growth of a variety of streptococcal species. To explore the mechanistic basis of this interaction, we performed a genetic screen to identify mutants of Streptococcus sanginuis SK36 whose growth was no longer enhanced by P. aeruginosa PAO1. Mutations in the zinc uptake systems of S. sanguinis SK36 reduced growth of these strains by 1 to 3 logs compared to that of wild-type S. sanguinis SK36 when grown in coculture with P. aeruginosa PAO1, and exogenous zinc (0.1 to 10 μM) rescued the coculture defect of zinc uptake mutants of S. sanguinis SK36. Zinc uptake mutants of S. sanguinis SK36 had no obvious growth defect in monoculture. Consistent with competition for zinc driving coculture dynamics, S. sanguinis SK36 grown in coculture with P. aeruginosa showed increased expression of zinc uptake genes compared to that of S. sanguinis grown alone. Strains of P. aeruginosa PAO1 defective in zinc transport also supported ∼2-fold more growth by S. sanguinis compared to that in coculture with wild-type P. aeruginosa PAO1. An analysis of 118 CF sputum samples revealed that total zinc levels varied from ∼5 to 145 μM. At relatively low zinc levels, Pseudomonas and Streptococcus spp. were found in approximately equal abundance; at higher zinc levels, we observed a decline in relative abundance of Streptococcus spp., perhaps as a result of increasing zinc toxicity. Together, our data indicate that the relative abundances of these microbes in the CF airway may be impacted by zinc levels. IMPORTANCE Polymicrobial infections in CF cases likely impact patient health, but the mechanism(s) underlying such interactions is poorly understood. Here, we show using an in vitro model system that interactions between Pseudomonas and Streptococcus are modulated by zinc availability, and clinical data are consistent with this model. Together with previous studies, our work supports a role for metal homeostasis as a key factor driving microbial interactions.

scholarly journals γ-Glutamyl Spermine Synthetase PauA2 as a Potential Target of Antibiotic Development against Pseudomonas aeruginosa

2012 ◽  
Vol 56 (10) ◽  
pp. 5309-5314 ◽  
Author(s):  
Xiangyu Yao ◽  
Congran Li ◽  
Jianmei Zhang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Serum ◽  
Parental Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Strong Support ◽  
Uptake System ◽  
Wild Type ◽  
Content Type ◽  
Antibiotic Development

ABSTRACTPolyamines are absolute requirements for cell growth. When in excess,Pseudomonas aeruginosapossesses six γ-glutamylpolyamine synthetases (GPSs) encoded by thepauA1-pauA7genes to initiate polyamine catabolism. Recently, thepauA2mutant was reported to lose the capability to grow on spermine (Spm) and spermidine (Spd) as sole carbon and nitrogen sources. Although this mutant grew normally in defined minimal medium and LB broth, growth was completely abolished by the addition of Spm or Spd. These two compounds exert a bactericidal effect (Spm > Spd) on the mutants as demonstrated by MIC measurements (over 500-fold reduction) and time-killing curves. Spm toxicity in thepauA2mutant was attenuated when the major uptake system was further deleted from the strain, suggesting cytoplasmic targets of toxicity. In addition, the synergistic effect of Spm and carbenicillin in the wild-type strain PAO1 was diminished in mutants without functional PauA2. Furthermore, Spm MIC was reduced by 8-fold when the Spm uptake system was deleted from the wild-type strain, suggesting a second target of Spm toxicity in the periplasm. Experiments were also conducted to test the hypothesis that native Spm and Spd in human serum may be sufficient to kill thepauA2mutant. Growth of the mutant was completely inhibited by 40% (vol/vol) human serum, whereas the parental strain required 80%. Colony counts indicated that the mutant but not the parent was in fact killed by human plasma. In addition, carbenicillin MIC against the mutant was reduced by 16-fold in the presence of 20% human serum while that of the parental strain remained unchanged. Taking PauA2 as the template, sequence comparison indicates that putative PauA2 homologues are widespread in a variety of Gram-negative bacteria. In summary, this study reveals the importance of GPS in alleviation of polyamine toxicity when in excess, and it provides strong support to the feasibility of GPS as a molecular target for new antibiotic development.

scholarly journals Impact of Two-Component Regulatory Systems PhoP-PhoQ and PmrA-PmrB on Colistin Pharmacodynamics in Pseudomonas aeruginosa

2012 ◽  
Vol 56 (6) ◽  
pp. 3453-3456 ◽  
Author(s):  
Neang S. Ly ◽  
Jenny Yang ◽  
Jurgen B. Bulitta ◽  
Brian T. Tsuji
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pharmacodynamic Model ◽  
Maximal Effect ◽  
Wild Type ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Subinhibitory Concentrations

ABSTRACTThein vitropharmacodynamics of colistin againstPseudomonas aeruginosaPAO1 wild-type and isogenic knockout strains ofphoPandpmrAwere evaluated. Colistin killing at subinhibitory concentrations was greater against thephoPandpmrAmutants than the wild type within the first 8 h: the concentration that results in 50% of maximal effect (EC50) of thepmrAmutant (0.413 mg/liter) was less than that of the wild type (0.718 mg/liter) (P< 0.05). Anin vitropharmacodynamic model simulating human colistin regimens displayed initial killing followed by regrowth in thephoPmutant and gradual regrowth in thepmrAmutant and wild type.

scholarly journals MrkD1Pfrom Klebsiella pneumoniae Strain IA565 Allows for Coexistence with Pseudomonas aeruginosa and Protection from Protease-Mediated Biofilm Detachment

2013 ◽  
Vol 81 (11) ◽  
pp. 4112-4120 ◽  
Author(s):  
Brandon M. Childers ◽  
Tricia A. Van Laar ◽  
Tao You ◽  
Steven Clegg ◽  
Kai P. Leung
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Chronic Wounds ◽  
Chronic Wound ◽  
Single Species ◽  
Wild Type ◽  
Content Type ◽  
Biofilm Model ◽  
Essential Components

ABSTRACTBiofilm formation and persistence are essential components for the continued survival of pathogens inside the host and constitute a major contributor to the development of chronic wounds with resistance to antimicrobial compounds. Understanding these processes is crucial for control of biofilm-mediated disease. Though chronic wound infections are often polymicrobial in nature, much of the research on chronic wound-related microbes has focused on single-species models.Klebsiella pneumoniaeandPseudomonas aeruginosaare microbes that are often found together in wound isolates and are able to form stablein vitrobiofilms, despite the antagonistic nature ofP. aeruginosawith other organisms. Mutants of theK. pneumoniaestrain IA565 lacking the plasmid-bornemrkD1Pgene were less competitive than the wild type in anin vitrodual-species biofilm model withP. aeruginosa(PAO1). PAO1 spent medium inhibited the formation of biofilm ofmrkD1P-deficient mutants and disrupted preestablished biofilms, with no effect on IA565 and no effect on the growth of the wild type or mutants. A screen using a two-allele PAO1 transposon library identified the LasB elastase as the secreted effector involved in biofilm disruption, and a purified version of the protein produced results similar to those with PAO1 spent medium. Various other proteases had a similar effect, suggesting that the disruption of themrkD1Pgene causes sensitivity to general proteolytic effects and indicating a role for MrkD1Pin protection against host antibiofilm effectors. Our results suggest that MrkD1Pallows for competition ofK. pneumoniaewithP. aeruginosain a mixed-species biofilm and provides defense against microbial and host-derived proteases.

scholarly journals Pathoadaptive Alteration of Salmonella Biofilm Formation in Response to the Gallbladder Environment

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Michael R. Neiger ◽  
Juan F. González ◽  
Geoffrey Gonzalez-Escobedo ◽  
Harkness Kuck ◽  
Peter White ◽  
...  
Keyword(s):  
Mouse Model ◽  
Typhoid Fever ◽  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Content Type ◽  
Chronic Carriage

ABSTRACT Typhoid fever, a human-specific disease, is primarily caused by the pathogen Salmonella enterica serovar Typhi. It is estimated that 3 to 5% of people infected with typhoid fever become chronic carriers. Studies have demonstrated that a mechanism of chronic carriage involves biofilm formation on gallstone surfaces. In the course of a previous study using a chronic carriage mouse model, a Salmonella enterica serovar Typhimurium isolate was recovered from a mouse gallstone that exhibited a 2-fold increase in biofilm formation over the wild type. In order to identify the gene(s) responsible for the phenotype, the genomic sequences of this isolate and others were determined and compared. These sequences identified single nucleotide polymorphisms (SNPs) in 14 genes. Mutations in the most promising candidates, envZ and rcsB, were created, but neither showed increased biofilm-forming ability separately or in combination. The hyperbiofilm isolate did, however, present variations in cellular appendages observable using different techniques and a preferential binding to cholesterol. The isolate was also examined for systemic virulence and the ability to colonize the gallbladder/gallstones in a mouse model of chronic infection, demonstrating a systemic virulence defect and decreased gallbladder/gallstone colonization. Finally, to determine if the appearance of hyperbiofilm isolates could be replicated in vitro and if this was a common event, wild-type Salmonella spp. were grown long term in vitro under gallbladder-mimicking conditions, resulting in a high proportion of isolates that replicated the hyperbiofilm phenotype of the original isolate. Thus, Salmonella spp. acquire random mutations under the gallbladder/gallbladder-simulating conditions that may aid persistence but negatively affect systemic virulence. IMPORTANCE Chronic carriers are the main reservoirs for the spread of typhoid fever in regions of endemicity. Salmonella Typhi forms biofilms on gallstones in order to persist. A strain with enhanced biofilm-forming ability was recovered after a nine-month chronic-carriage mouse study. After sequencing this strain and recreating some of the mutations, we could not duplicate the phenotype. The isolate did show a difference in flagella, a preference to bind to cholesterol, and a systemic virulence defect. Finally, gallbladder conditions were simulated in vitro. After 60 days, there was a 4.5-fold increase in hyperbiofilm isolates when a gallstone was present. These results indicate that Salmonella spp. can undergo genetic changes that improve persistence in gallbladder albeit at the cost of decreased virulence.

scholarly journals Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

2013 ◽  
Vol 82 (2) ◽  
pp. 544-556 ◽  
Author(s):  
W. Brian Whitaker ◽  
Gary P. Richards ◽  
E. Fidelma Boyd
Keyword(s):  
Mouse Model ◽  
Sigma Factor ◽  
Vibrio Parahaemolyticus ◽  
Wild Type ◽  
Carbon Utilization ◽  
Content Type ◽  
Wide Range ◽  
Competition Assays

ABSTRACTVibrio parahaemolyticusis the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation inrpoN(VP2670) inV. parahaemolyticusRIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effectsin vivousing a streptomycin-treated mouse model of colonization. We confirmed that deletion ofrpoNrenderedV. parahaemolyticusnonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. Inin vivocompetition assays between therpoNmutant and a wild-type RIMD2210633 strain marked with the β-galactosidase genelacZ(WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that therpoNmutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as therpoNmutant, which suggested that lack of motility is not the sole cause of the fitness effect. In anin vitrogrowth competition assay in mouse intestinal mucus, therpoNmutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ΔrpoNmutant than in the wild type. These data suggest that inV. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization.

scholarly journals An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Brian M. Peters ◽  
Arturo Luna-Tapia ◽  
Hélène Tournu ◽  
Jeffrey M. Rybak ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Vaginal Candidiasis ◽  
Azole Resistance ◽  
Endosomal Trafficking ◽  
Wild Type ◽  
Content Type ◽  
Azole Antifungals

ABSTRACT We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

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