Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments

Pestrak, Matthew J.; Chaney, Sarah B.; Eggleston, Heather; Dellos-Nolan, Sheri; Dixit, Sriteja; Mathew-Steiner, Shomita S.; Roy, Sashwati; Parsek, Matthew R.; Sen, Chandan K.; Wozniak, Daniel J.

doi:10.1371/journal.ppat.1006842

Cited by 95 publications

(88 citation statements)

References 76 publications

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“…Additionally, Staudinger et al (16) demonstrated that host factors associated with the CF lung environment promote bacterial aggregation, which in turn contributes to reduced killing by neutrophils and the host defense oxidants, H 2 O 2 and HOCl (15,16). Aggregation may also be associated with recurrent inflammation in chronic P. aeruginosa infections, as well as with protection from antimicrobial products (17,18,35,42).…”

Section: Discussionmentioning

confidence: 99%

“…Next, we investigated whether the tolerance of aggregated bacteria to gentamicin was due to protection conferred by the physiology of bacteria within the aggregates. With the use of established methodology (35), the aggregates were physically disrupted by forcing them through a 22-gauge needle several times prior to antibiotic treatment. Interestingly, disruption of aggregated PAO1 WT bacteria did not substantially increase susceptibility of bacteria to gentamicin (Fig.…”

Section: P Aeruginosa (Pao1) Flagella Are Dispensable For Formation mentioning

confidence: 99%

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Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

Demirdjian

Sanchez

Hopkins

et al. 2019

Appl Environ Microbiol

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Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections. IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

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Section: Discussionmentioning

confidence: 99%

Section: P Aeruginosa (Pao1) Flagella Are Dispensable For Formation mentioning

confidence: 99%

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

Demirdjian

Sanchez

Hopkins

et al. 2019

Appl Environ Microbiol

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“…In P. aeruginosa , increased c-di-GMP, among many responses, leads to overproduction of exopolysaccharides, Psl and Pel, and matrix proteins 12 , 13 . As a result, RSCVs have hyper-biofilm phenotypes 13 , 14 , increased tolerance to antimicrobials 10 , and enhanced resistance to immunity 15 , 16 . P. aeruginosa RSCVs also evolve from in vitro grown biofilms 17 , 18 , suggesting that there is strong selection for ecological diversification in both in vivo and in vitro biofilms.…”

Section: Introductory Paragraphmentioning

confidence: 99%

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

Gloag

Marshall

Snyder

et al. 2018

Preprint

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Running title: Evolution of P. aeruginosa RSCVs in a chronic infection model 22Author Contributions: ESG and CWM performed the experimental work. JSH 23 performed the colony PCR. SBC infected and sampled the porcine burn wounds. DS 24 generated the sequence library. MW and GRL quantified the strain frequency in the 25 wounds. ESG, CWM, MW, VSC, and DJW conceptualized the project and wrote the 26 manuscript. 27Chronic infections are those that persist despite extensive treatment. These 55 persistent infections are often attributed to difficult to eradicate biofilms, which are 56 communities of adhered microorganisms encased in an extracellular polymeric 57 substance (EPS) 1,2 . Complicating chronic infections is the high likelihood that 58 bacterial populations adaptively evolve, producing persistent phenotypes with 59 increased fitness. 60 61One of the most understood bacterial adaptive responses to chronic infection is that 62of Pseudomonas aeruginosa to the cystic fibrosis (CF) lung 3 . CF patients exhibit 63 mucus accumulation, where P. aeruginosa biofilms commonly colonize the mucus 64 lining and establish persistent pulmonary infections 4-6 . Evolved phenotypic variants 65 of this organism are routinely isolated from CF patient sputum samples, and several 66 of these variants are often associated with worsening patient prognosis 7 . Of 67 particular interest are the rugose small-colony variants (RSCVs), which are isolated 68 from up to 50% of P. aeruginosa-positive CF sputum samples 8,9 . When isolated, 69 their frequencies range drastically between 0.1 − 100% 9,10 . In contrast there is little-70 to-no reports of the frequency of P. aeruginosa adapted variants from other chronic 71 infections, such as chronic wound infections. 72 73 Common to RSCVs are mutations in pathways that lead to elevated cyclic 74 diguanylate monophosphate (c-di-GMP) 7 . C-di-GMP is a messenger molecule that 75 signals the transition from planktonic to biofilm lifestyle in many bacteria 11 . In P. 76 aeruginosa, increased c-di-GMP, among many responses, leads to overproduction of 77 exopolysaccharides, Psl and Pel, and matrix proteins 12,13 . As a result, RSCVs have 78 hyper-biofilm phenotypes 13,14 , increased tolerance to antimicrobials 10 , and enhanced 79 resistance to immunity 15,16 . P. aeruginosa RSCVs also evolve from in vitro grown 80 biofilms 17,18 , suggesting that there is strong selection for ecological diversification in 81 both in vivo and in vitro biofilms. 82 defined by a matte, rugose colony morphology that stained intensely by the two dyes, 132 indicating exopolysaccharide overproduction, were isolated from all three timepoints 133 (Fig 2A). Two RSCV sub-populations were observed; one that had a pink, rugose 134 phenotype and a second that had an orange, textured phenotype (Fig 2A). 135 136The RSCV abundance in the wounds was quantified by expressing their frequency 137 as a percentage of the total P. aeruginosa burden. The RSCV frequency was low in 138

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“…Further analysis of DC-SIGN and MR binding to P. aeruginosa biofilms using ELISA-based assays identified binding of DC-SIGN to biofilms generated by the Psl-deficient mutant Δ wsp F Δ psl (16)(Table 1); this mutant is not expected to contain mannose-rich carbohydrates. The Δ wspF background confers constitutive high levels of cyclic-di-GMP, overproduces Psl and promotes biofilm formation; a phenotype that resembles that of small rough colony variants found during chronic infection (17). MR-CTLD4-7 displays reduced binding to Δ wsp F Δ psl biofilms indicating that the psl operon is likely responsible for the generation of MR-CTLD4-7 ligands (Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

Pseudomonas aeruginosabiofilms display carbohydrate ligands for CD206 and CD209 that interfere with their receptor function

Singh

Almuhanna

Alshahrani

et al. 2020

Preprint

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40Bacterial biofilms represent a challenge to the healthcare system because of their resilience 41 against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded 42 in an extracellular polymeric substance (EPS) composed of carbohydrate polymers, nucleic 43 acids and proteins. Carbohydrates within P. aeruginosa biofilms include neutral and mannose-44 rich Psl, and cationic Pel composed of N-acetyl-galactosamine and N-acetyl-glucosamine. 45 Here we show that P. aeruginosa biofilms display ligands for the C-type lectin receptors 46 mannose receptor (MR, CD206) and Dendritic Cell-Specific Intercellular adhesion molecule-47 61 Author Summary 62Selective engagement of pattern recognition receptors during infection guides the decision-63 making process during induction of immune responses. This work identifies mannose-rich 64 carbohydrates within bacterial biofilms as novel molecular patterns associated with bacterial 65 infections. P. aeruginosa biofilms and biofilm-derived carbohydrates bind two important lectin 66 receptors, MR (CD206) and DC-SIGN (CD209), involved in recognition of self and immune 67 evasion. Abundance of MR and DC-SIGN ligands in the context of P. aeruginosa biofilms 68 could impact immune responses and promote chronic infection. 69 70 Results 107 P. aeruginosa biofilms display DC-SIGN and MR ligands 108The mannose-rich nature of carbohydrates produced by P. aeruginosa biofilms (9) suggested 109 the possibility of immune mannose-specific lectins recognising these structures. We tested 110 whether the lectins MR and DC-SIGN bound P. aeruginosa biofilms by analysing the 111 interaction of recombinant Fc chimeric molecules DC-SIGN-Fc and MR-CTLD4-7-Fc (14) to 112 biofilms generated in 96 well plates as described in Materials and Methods. Both DC-SIGN 113 and MR-CTLD4-7 bound to P. aeruginosa PAO1 biofilms and DC-SIGN displayed higher 114 binding compared to MR ( Figure 1A). Inhibition assays using selected monosaccharides, 115confirmed that MR and DC-SIGN binding to PAO1 biofilms is carbohydrate-dependent and 116 preferentially inhibited by mannose and fucose, in agreement with the sugar specificity 117 previously shown for both lectins ( Figure 1A), and Ca 2+ -dependent ( Figure 1B), as expected 118 for these C-type lectins (11, 12). Recognition by MR and DC-SIGN was not restricted to 119 biofilms generated by PAO1 and extends to biofilms generated by wound clinical isolates (CW 120 2, CW3, CW5-B, CW5-S, CW6 and CW7). This strain collection comprised isolates collected 121 from bone (CW2 and CW5-B), wound tissue (CW3, CW6 and CW7) and blood (CW5-S) and 122 two serotypes (13)(CW2, CW3, CW5 and CW6 are serotype O6) and CW7 is part of the 123 serotype O5 cluster, which includes O5, O18, and O20 serotypes. Sequence analysis 124 confirmed presence of the psl operon (8) in all isolates although point mutations could alter 125 the levels and structure of the carbohydrate ( Figure S1). DC-SIGN and MR-CTLD4-126 7 binding to biofilms formed by clinical isolates follows a simila...

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Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments

Cited by 95 publications

References 76 publications

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

Pseudomonas aeruginosabiofilms display carbohydrate ligands for CD206 and CD209 that interfere with their receptor function

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