524: Transcriptomic analysis on the effects of cysteamine in synthetic sputum on the virulence and metabolism of Pseudomonas aeruginosa and Burkholderia cenocepacia

ABSTRACTThe treatment ofStenotrophomonas maltophiliainfection with β-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded β-lactamases. Here, we show that these β-lactamase–packed OMVs are capable of establishing extracellular β-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating speciesPseudomonas aeruginosaandBurkholderia cenocepacia.

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Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag

Pharmaceuticals ◽

10.3390/ph14100956 ◽

2021 ◽

Vol 14 (10) ◽

pp. 956

Author(s):

Ana Margarida Pereira ◽

André da Costa ◽

Simoni Campos Dias ◽

Margarida Casal ◽

Raul Machado

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Klebsiella Pneumoniae ◽

Antimicrobial Peptides ◽

Formic Acid ◽

Cleavage Site ◽

Burkholderia Cenocepacia ◽

High Recovery ◽

Temperature Cycles ◽

Acid Cleavage

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.

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Transcriptomic Analysis Reveals the Dependency of Pseudomonas aeruginosa Genes for Double-Stranded RNA Bacteriophage phiYY Infection Cycle

iScience ◽

10.1016/j.isci.2020.101437 ◽

2020 ◽

Vol 23 (9) ◽

pp. 101437

Author(s):

Qiu Zhong ◽

Lan Yang ◽

Linlin Li ◽

Wei Shen ◽

Yang Li ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcriptomic Analysis ◽

Infection Cycle ◽

Double Stranded Rna

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Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

Infection and Immunity ◽

10.1128/iai.01876-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4729-4745 ◽

Cited By ~ 57

Author(s):

Ute Schwab ◽

Lubna H. Abdullah ◽

Olivia S. Perlmutt ◽

Daniel Albert ◽

C. William Davis ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Single Cells ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Test Medium ◽

Fermentation Products ◽

Content Type

ABSTRACTThe localization ofBurkholderia cepaciacomplex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection withPseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc andP. aeruginosabacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallelin vitroexperiments examined the growth of two Bcc species,Burkholderia cenocepaciaandBurkholderia multivorans, in environments similar to those occupied byP. aeruginosain the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast,P. aeruginosawas identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions withP. aeruginosain an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions,B. cenocepaciaandB. multivoransused fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence ofP. aeruginosain vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade aP. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibitP. aeruginosabiofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.

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Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI Secretion System-Mediated Predation by the Burkholderia cepacia Complex

10.1101/2020.04.10.036012 ◽

2020 ◽

Author(s):

Andrew I Perault ◽

Courtney E Chandler ◽

David A Rasko ◽

Robert K Ernst ◽

Matthew C Wolfgang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Secretion System ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Dependent Manner ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Type Vi Secretion

SUMMARYPseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of individuals with cystic fibrosis (CF). While Pa can initiate long-term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both Pa and Bcc use type VI secretion systems (T6SS) to mediate interbacterial competition. Here, we show that Pa isolates from teenage/adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054 (BcAU1054) in a T6SS-dependent manner. The genomes of susceptible Pa isolates harbor T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant. Moreover, seven of eight Bcc strains outcompeted Pa strains isolated from the same patients. Our findings suggest that certain mutations that arise as Pa adapts to the CF lung abrogate T6SS activity, making Pa and its human host susceptible to potentially fatal Bcc superinfection.

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Iron Availability Influences Aggregation, Biofilm, Adhesion and Invasion of Pseudomonas Aeruginosa and Burkholderia Cenocepacia

International Journal of Immunopathology and Pharmacology ◽

10.1177/039463200501800407 ◽

2005 ◽

Vol 18 (4) ◽

pp. 661-670 ◽

Cited By ~ 63

Author(s):

F. Berlutti ◽

C. Morea ◽

A. Battistoni ◽

S. Sarli ◽

P. Cipriani ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Iron Concentration ◽

Burkholderia Cenocepacia ◽

Iron Availability ◽

Opportunistic Pathogens ◽

Free Living ◽

Healthy Humans ◽

Systemic Spread ◽

Epithelial Barriers

Pseudomonas aeruginosa and Burkholderia cenocepacia are predominant opportunistic pathogens in cystic fibrosis (CF) patients. In healthy humans the lower respiratory tract as well as all mucosa, contains a very low free iron concentration (10−18 M), while in CF patients' sputum iron concentration is very high, showing a median value of 63×10−6 M. Accumulation of catalytic reactive iron heavily contributes to subsequent clinical complications in the lung disorders by the production of reactive oxygen species and increases bacterial growth and virulence. The data reported in this study indicate that low iron concentration (Fe3+1 μM) induced free-living forms and motility both in P. aeruginosa and B. cenocepacia, while high iron concentrations (Fe3+ 10 and 100 μM) stimulated aggregation and biofilm formation already in the fluid phases, so demonstrating that aggregation and biofilm formation are positively iron-modulated in these bacteria. Moreover, the different morphological forms (free-living, aggregates and biofilm) showed different capabilities of adhering and invading the bronchial cell line A549. P. aeruginosa PAO1 aggregates, and mostly biofilm, exerted the highest adhesion efficiency, while B. cenocepacia PV1 aggregates or biofilm the lowest. A significant reduction in invasion efficiency by P. aeruginosa biofilm and a significant increase in cell internalization by B. cenocepacia biofilm has been reported. Therefore, the iron availability is an important signal to which P. aeruginosa and B. cenocepacia counteract by leaving the motile free-living forms and entering into a new lifestyle, i.e. biofilm. These data could contribute to explain that the iron-overload of the sputum of CF patients, inducing nonmotile forms, aggregates and biofilm, may facilitate penetration of host epithelial barriers contributing to the establishment of infection, colonization, persistence and systemic spread of these opportunistic pathogens.

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Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.070672-0 ◽

2014 ◽

Vol 160 (2) ◽

pp. 261-269 ◽

Cited By ~ 28

Author(s):

Ameer Elfarash ◽

Jozef Dingemans ◽

Lumeng Ye ◽

Ahmed Amir Hassan ◽

Michael Craggs ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Receptor Binding ◽

Protein Product ◽

Binding Domain ◽

Burkholderia Cenocepacia ◽

Receptor Binding Domain ◽

Amino Acid Residues ◽

Gene Encoding ◽

Siderophore Receptors ◽

Transposon Mutants

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

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