Date Published: December 27, 2018
Publisher: Public Library of Science
Author(s): Hanna Ostapska, P. Lynne Howell, Donald C. Sheppard, Deborah A. Hogan.
The production of biofilms is a common strategy used by many microorganisms during infection. Exopolysaccharides are a major component of the extracellular biofilm matrix serving to anchor organisms to surfaces, forming the structural scaffold of the biofilm, and protecting organisms from damage by hostile factors such as antibiotics and host immune defenses (Fig 1). Biochemical and genetic studies of biofilm exopolysaccharide synthesis have revealed that production of N-acetyl hexosamine-containing exopolysaccharides is one strategy used by diverse pathogens to facilitate biofilm formation and virulence. Following polymerization and extracellular extrusion by membrane embedded glycosyl transferases ([HexNAc]n + nucleotide-HexNac → [HexNAc]n+1 + nucleotide), these glycans then undergo postsynthetic enzymatic deacetylation ([HexNAc)n → HexN-[HexNAc]n-1 + acetyl group) to render them cationic. Deacetylation is critical for the function of these glycans in biofilm formation and host–pathogen interactions. This Pearl explores the role of these deacetylated cationic exopolysaccharides within the biofilm matrix in microbial pathogenesis and resistance to antimicrobial agents, and their potential as antibiofilm therapeutic targets.
A wide range of medically important microbial species produce and secrete hexosamine-rich exopolysaccharides into their self-produced extracellular biofilm matrices (Table 1). The best studied example of these glycans is poly-β-1,6-N-acetylglucosamine (PNAG), a homopolymer of N-acetylglucosamine (GlcNAc) residues produced by a wide range of gram-positive and gram-negative pathogenic bacteria, including Staphylococcus spp., Yersinia pestis, Bordetella spp., and Escherichia coli [1–4]. The gram-negative opportunistic pathogen Pseudomonas aeruginosa produces several biofilm-associated exopolysaccharides, including the linear heteropolymer Pel, composed of GlcNAc and N-acetyl galactosamine (GalNAc), whereas the gram-positive organism Listeria monocytogenes produces a β-1,4-linked N-acetylmannosamine polysaccharide decorated with terminal α-1,6-linked galactose (Gal) residues [5,6]. More recently, biofilm formation by the opportunistic filamentous fungal pathogen Aspergillus fumigatus was found to be dependent on galactosaminogalactan (GAG), a heteropolymer composed of α-1,4-linked GalNAc and Gal residues .
Deacetylation plays an important role in the synthesis, transport, and localization of many of these exopolysaccharides. Mutants of E. coli, L. monocytogenes, P. aeruginosa, Y. pestis, Staphylococcus spp., Bordetella bronchiseptica, and A. fumigatus deficient in their respective exopolysaccharide deacetylase were found to lack detectable cell-wall–associated polysaccharide [6,8,10–12,15]. In the case of L. monocytogenes and P. aeruginosa, deacetylation of their polymers seems to be required for polymer synthesis, whereas in E. coli, loss of deacetylase activity results in retention of immature polymer in the periplasm [6,11,17]. Studies of Staphylococcus epidermidis and A. fumigatus have demonstrated that the loss of exopolysaccharide deacetylation in these species results in the production of a fully acetylated glycan that is shed in the culture supernatant and does not adhere to the cell wall. This observation suggests that cationic glycans likely adhere to anionic components of the bacterial and fungal cell wall through charge.
Mutant organisms deficient in the production of GAG and PNAG exhibit attenuated virulence in mouse or invertebrate models of infection [1,3,7,10,19,20]. These experiments suggest that the production of cationic, adhesive exopolysaccharides also provides protection against detection and elimination by elements of the host immune system. Adhesion of exopolysaccharides to the microbial cell wall can conceal cell-surface pathogen-associated molecular patterns from immune recognition by the innate immune system. This phenomenon has been best studied in A. fumigatus, for which deletion of the deacetylase Agd3 leads to greater recognition of β-glucans on the surface of hyphae by pattern recognition receptor Dectin-1 . Similar findings were observed with the loss of PNAG deacetylation in S. epidermidis, resulting in a mutant prone to being avidly phagocytosed by human neutrophils . PNAG has also been shown to inhibit complement deposition on the cell surface of Bordetella pertussis . The presence of Pel in P. aeruginosa biofilms provides protection from killing by the leukocyte-like cell line HL-60, although the mechanism by which this occurs has not been defined .
Cationic exopolysaccharides can also mediate resistance to antimicrobial agents through repulsion or sequestration of these molecules. PNAG production increases the biofilm resistance of Aggregatibacter actinomycetemcomitans to the cationic detergent cetylpyridinium chloride and protects S. epidermidis against microbicidal action of glycopeptide antibiotics, such as vancomycin [9,24]. The production of GAG by A. fumigatus limits intracellular penetration of the hydrophobic antifungal posaconazole and reduces its activity . Degrading Pel polysaccharide within P. aeruginosa biofilms enhanced susceptibility to colistin, and disrupting the Pel operon resulted in enhanced susceptibility of P. aeruginosa to the aminoglycosides tobramycin and gentamicin [16,18]. The ability of Pel to enhance antimicrobial resistance may be strain or condition dependent, however, because other studies have reported that disruption of the PelA deacetylase failed to alter susceptibility to tobramycin or ciprofloxacin . It has been suggested that Pel polysaccharide may enhance antimicrobial resistance through interacting with and anchoring other macromolecules such as anionic extracellular DNA (eDNA) within P. aeruginosa biofilms, which in turn can act to sequester or repel antimicrobials and thereby prevent access to intracellular targets . Although similar interactions of other cationic polysaccharides with eDNA have not been reported, it is likely for these cationic exopolysaccharides to have similar roles within their respective biofilms. Together, these observations suggest that the deacetylation of polymers also actively contributes to protection of their respective organisms against antimicrobials.
Studies of the biosynthetic pathways governing the production of deacetylated exopolysaccharides have suggested that these pathogens produce hydrolytic enzymes specific for cleavage of these polymers. These enzymes include Sph3 to cleave A. fumigatus GAG, the hydrolase domain of PelA to cleave P. aeruginosa Pel, and PssZ to cleave L. monocytogenes exopolysaccharide (EPS) [6,14,26]. Cross-species activity of hydrolytic enzymes has also been demonstrated. Dispersin B (DspB) of A. actinomycetemcomitans and NghA of Y. pseudotuberculosis cleave PNAG β-1,6-linked GlcNAc polymers and residues, respectively, and the P. aeruginosa PelA hydrolase cleaves fungal GAG [14,27,28].
Although our understanding of the biosynthetic pathways governing the synthesis of deacetylated exopolysaccharides has expanded greatly, there are many unanswered questions about their functions in host–pathogen interactions. For example, studies of A. fumigatus GAG suggest that purified fractions of this glycan can directly modulate host immune responses, although the receptors and signaling pathways governing this process remain unknown [35,36]. These and similar studies in other organisms await robust protocols for the purification of deacetylated exopolysaccharides or the production of synthetic oligosaccharides derived from these glycans. In addition, the function of deacetylated exopolysaccharides has been largely studied in the context of single-species biofilms. However, as these organisms typically exist in polymicrobial environments, the potential for exopolysaccharides to play cooperative roles in multispecies biofilms also needs to be explored.