Proteomic, Microarray, and Signature-Tagged Mutagenesis Analyses of Anaerobic Pseudomonas aeruginosa at pH 6.5, Likely Representing Chronic, Late-Stage Cystic Fibrosis Airway Conditions

ABSTRACT Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of ∼6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant “anaerobiome” or “proteogenome” of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO3 − and NO2 − are those encoding Pf1 bacteriophage. With anaerobic NO2 −, the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO3 − and NO2 − respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO3 − and NO2 − respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO3 − and NO2 − respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.

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Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway

Pulmonary Pharmacology & Therapeutics ◽

10.1016/j.pupt.2007.12.001 ◽

2008 ◽

Vol 21 (4) ◽

pp. 595-599 ◽

Cited By ~ 170

Author(s):

Sophie Moreau-Marquis ◽

Bruce A. Stanton ◽

George A. O’Toole

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Cystic Fibrosis Airway

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Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung

Journal of Bacteriology ◽

10.1128/jb.00119-09 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3492-3503 ◽

Cited By ~ 227

Author(s):

Melissa Starkey ◽

Jason H. Hickman ◽

Luyan Ma ◽

Niu Zhang ◽

Susan De Long ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Intracellular Signaling ◽

Tricarboxylic Acid Cycle ◽

Gene Clusters ◽

Wild Type ◽

Phenotypic Analysis ◽

Small Colony Variants ◽

Airway Infections ◽

Small Colony

ABSTRACT Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.

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A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0606428103 ◽

2006 ◽

Vol 103 (48) ◽

pp. 18131-18136 ◽

Cited By ~ 142

Author(s):

H. Matsui ◽

V. E. Wagner ◽

D. B. Hill ◽

U. E. Schwab ◽

T. D. Rogers ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Cystic Fibrosis Airway ◽

Physical Linkage

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Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dku357 ◽

2014 ◽

Vol 70 (1) ◽

pp. 160-166 ◽

Cited By ~ 25

Author(s):

S. Moreau-Marquis ◽

B. Coutermarsh ◽

B. A. Stanton

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Cystic Fibrosis Airway

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Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

10.1101/2020.02.25.964320 ◽

2020 ◽

Cited By ~ 2

Author(s):

Wai Leong ◽

Carla Lutz ◽

Jonathan Williams ◽

Yan Hong Poh ◽

Benny Yeo Ken Yee ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Late Stage ◽

Early Stage ◽

Acanthamoeba Castellanii ◽

Opportunistic Pathogen ◽

Human Neutrophils ◽

Phenotypic Traits ◽

Chronic Infections

AbstractThe opportunistic pathogen, Pseudomonas aeruginosa, is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa, when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii, for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans, was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types.Author SummaryPseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions.

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Adaptation to an amoeba host leads to Pseudomonas aeruginosa isolates with attenuated virulence

Applied and Environmental Microbiology ◽

10.1128/aem.02322-21 ◽

2022 ◽

Author(s):

Wai Leong ◽

Wee Han Poh ◽

Jonathan Williams ◽

Carla Lutz ◽

M. Mozammel Hoque ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Late Stage ◽

Early Stage ◽

Opportunistic Pathogen ◽

Phenotypic Traits ◽

Nucleotide Polymorphisms ◽

Chronic Infections ◽

The Impact

The opportunistic pathogen Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when co-incubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and non-adapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoeba as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoeba resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. Importance Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by bacterial predators, e.g. protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examine the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa . We show that long term co-incubation with protozoa resulted in mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures amongst host cell types as well as similar adaptation strategies.

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