Antibodies to PhnD Inhibit Staphylococcal Biofilms

Abstract

Biofilm formation on central lines or peripheral catheters is a serious threat to patient well-being. Contaminated vascular devices can act as a nidus for bloodstream infection and systemic pathogen dissemination. Staphylococcal biofilms are the most common cause of central-line-associated bloodstream infections, and antibiotic resistance makes them difficult to treat. As an alternative to antibiotic intervention, we sought to identify anti-staphylococcal biofilm targets for the development of a vaccine or antibody prophylactic. A screening strategy was devised using a microfluidic system to test antibody-mediated biofilm inhibition under biologically relevant conditions of shear flow. Affinity-purified polyclonal antibodies to target antigen PhnD inhibited both Staphylococcus epidermidis and S. aureus biofilms. PhnD-specific antibodies blocked biofilm development at the initial attachment and aggregation stages, and deletion of phnD inhibited normal biofilm formation. We further adapted our microfluidic biofilm system to monitor the interaction of human neutrophils with staphylococcal biofilms and demonstrated that PhnD-specific antibodies also serve as opsonins to enhance neutrophil binding, motility, and biofilm engulfment. These data support the identification of PhnD as a lead target for biofilm intervention strategies performed either by vaccination or through passive administration of antibodies.

Disruption of staphylococcal aggregation protects against lethal lung injury

“…WT preincubated with a polyclonal antibody that inhibits PhnD binding (11), caused MA formation. However, in each case we could wash out the MAs with alveolar buffer microinstillation ( Figure 2F, third and fourth bars from left), indicating that the clusters were not stable.…”

“…To evaluate bacterial proteins in MA formation, we considered the role of PhnD. Although PhnD is a component of the bacterial phosphonate ABC transporter (27,28), reports indicate that it may have also an adhesive function in vitro (11). Alveolar microinstillation of PhnD-deficient mutant USA300 (phnD -) (29), or USA300…”

“…We interpret that the MAs formed by PhnD-null USA300 were poorly organized and therefore unstable. Substrate-binding proteins of ABC transporters, such as PhnD (27,28), are known to play a role in bacterial adherence in vitro (11,53,54). However, underlying adherence-inducing mechanisms remain unclear.…”

“…Studies in vitro indicate that the bacteria may stabilize through biofilm formation (8,9), charge interactions (10), or PhnD, the substrate-binding protein of the bacterial ATP-binding cassette (ABC) transporter for phosphonates (11). Once stabilized on the alveolar epithelium, S. aureus may cause injury by metalloproteinase activation (12), cytokine receptors (6), necroptosis (13), and mitochondrial dysfunction (14).…”

Efflux as a Glutaraldehyde Resistance Mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa Biofilms

“…Phosphonate degradation, lipid metabolism, and polyamine biosynthesis have been reported to influence biofilm formation. For instance, antibodies against PhnD, the phosphonate ABC transporter substrate-binding protein, were reported to block initial attachment and aggregation (induced 2.5-fold in our study) (47). Similarly, a defect in polyamine biosynthesis has been shown to reduce planktonic and biofilm growth in several species that may be rescued with exogenous polyamine supplementation (48)(49)(50)(51)(52).…”