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

Abstract

Pseudomonas aeruginosa causes devastating infections in immunocompromised individuals. Once established, P. aeruginosa infections become incredibly difficult to treat due to the development of antibiotic tolerant, aggregated communities known as biofilms. A hyper-biofilm forming clinical variant of P. aeruginosa, known as a rugose small-colony variant (RSCV), is frequently isolated from chronic infections and is correlated with poor clinical outcome. The development of these mutants during infection suggests a selective advantage for this phenotype, but it remains unclear how this phenotype promotes persistence. While prior studies suggest RSCVs could survive by evading the host immune response, our study reveals infection with the RSCV, PAO1ΔwspF, stimulated an extensive inflammatory response that caused significant damage to the surrounding host tissue. In both a chronic wound model and acute pulmonary model of infection, we observed increased bacterial burden, host tissue damage, and a robust neutrophil response during RSCV infection. Given the essential role of neutrophils in P. aeruginosa-mediated disease, we investigated the impact of the RSCV phenotype on neutrophil function. The RSCV phenotype promoted phagocytic evasion and stimulated neutrophil reactive oxygen species (ROS) production. We also demonstrate that bacterial aggregation and TLR-mediated pro-inflammatory cytokine production contribute to the immune response to RSCVs. Additionally, RSCVs exhibited enhanced tolerance to neutrophil-produced antimicrobials including H2O2 and the antimicrobial peptide LL-37. Collectively, these data indicate RSCVs elicit a robust but ineffective neutrophil response that causes significant host tissue damage. This study provides new insight on RSCV persistence, and indicates this variant may have a critical role in the recurring tissue damage often associated with chronic infections.

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

“…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).…”

“…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.…”

ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

“…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.…”

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

“…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).…”