Interaction of polymicrobial biofilms with human immune cells

Multidrug resistance is causing redundancy of entire classes of antibiotics and making the end of the "antibiotic era", in which common infections and minor injuries can kill, a real possibility. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) are resistant to the biocidal action of antibiotics and there is an urgent need for the development of new antimicrobial options underpinned by a better understanding of the pathogenicity of ESKAPE organisms.

P. aeruginosa (PA) is a highly versatile environmental organism. PA flourishes in the hospital setting where it causes serious infections in immunocompromised and burn patients. PA infection is especially troublesome in Cystic Fibrosis (CF) patients where it is a major determinant of the irreversible loss of lung function and mortality. Breach of immune defences enables PA to cause intractable infections due to its intrinsic resistance to antibiotics and ability to form biofilms. Transition from planktonic to sessile growth and biofilm development are central to PA pathogenesis. There is an urgent need to better understand how the switch to a biofilm life style contributes to PA virulence and how immune cells detect and respond to these structures.

PA biofilms consist of an extracellular matrix containing two main carbohydrate polymers, Psl and Pel. Our recent results show that three major carbohydrate-binding (lectin) receptors expressed by immune cells, DC-SIGN (CD209), the mannose receptor (MR, CD206) and Dectin-2, interact with PA biofilms. Binding is calcium-dependent, inhibited by relevant sugars, and observed in biofilms formed by PAO1 and clinical isolates. DC-SIGN, MR and Dectin-2 ligands within biofilms showed differential distribution. Human monocyte-derived dendritic cells (huDCs) incubated with wells containing Psl+/Pel+, Psl+/Pel- and Psl-/Pel+ biofilms alongside wells containing bacteria non capable of biofilm formation Psl-/Pel- selectively produce IL-1B (4 and 18 h) and IL-23 (only 4 h) in response to biofilm-containing wells, with biofilms containing more Psl tending to induce less IL 23. Further, specific blockage of lectin receptors in huDCs alters their response to PA biofilms. These results suggest that biofilms do not just provide an effective barrier against immune attack but could also modulate immune cell activation in the context of PA infection through engagement of lectin receptors.

Many infections, such as pulmonary infections occurring in patients with CF, are polymicrobial in nature. Most children with CF are initially colonised with S. aureus in their airways. With time S. aureus is commonly replaced with PA, resulting in a period of co-infection. S. aureus can produce a multilayered biofilm embedded within a glycocalyx or slime layer. The glycocalix contains a polysaccharide antigen named polysaccharide intercellular antigen (PIA). PIA is composed of B-1,6-linked N-acetylglucosamine residues (80-85%) and an anionic fraction with a lower content of non-N-acetylated D-glucosaminyl residues that contains phosphate and ester-linked succinate (15-20%). No receptors for PIA have been described to date.

We hypothesise that mixed biofilms formed by PA and S. aureus will display altered carbohydrate production leading to differential engagement of lectin receptors and differential activation of human immune cells.

The specific aims of this project are (i) to investigate how PA and S. aureus mixed biofilms influence the activation of human immune cells (ii) and determine the contribution of lectin receptors to these responses.