Structural Diversity in the Type IV Pili of Multidrug-resistant Acinetobacter

Abstract

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating that A. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter. Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species.Type IV pili are extracellular adhesive appendages primarily comprising a single protein subunit, called the major pilin, which is assembled into a narrow (ϳ6 -9-nm) helical fiber of variable length (up to 2.5 m) (1). One or more other proteins, called minor pilins, are also incorporated into the fiber at low levels. All pilins contain an N-terminal signal sequence followed by an ϳ30-amino acid hydrophobic ␣-helix resembling a transmembrane domain (the ␣1-N domain). This is, in turn, followed by a soluble ϳ15-kDa globular domain referred to as the pilin headgroup; the hydrophobic helical regions are buried together in the center of the fiber, whereas portions of the C-terminal headgroup are exposed (2, 3).Type IV pili are found in both Gram-negative (4, 5) and Gram-positive (6 -8) bacteria as well as Archea (9). They are involved in a wide range of processes, including twitching motility (10), horizontal gene transfer (11), host-cell adhesion (12), and microcolony/biofilm formation (13). This functional diversity is reflected in the sequence of the pilin proteins that typically have little or no sequence identity beyond the hydrophobic portion of the N-terminal ␣-helix. This lack of sequence identity is apparent even in cases where there is high structural similarity.In contrast, within a given species, the minor pilins are typically well conserved. Only the major pilin is highly variable (14 -16) and then only in those regions left exposed in the assembled pilus (17). This sequence diversity may result from diversifying selection as a mechanism by which to avoid detection by the host immune system (18). However, such diversity can also be found in species whose life cycle is primarily environmental (19). Glycosylation is an additional source of variability in some type IV pilins; O-linked glycosylation has been observed in multiple strains of both Pseudomonas aeruginosa (20 -22) and Neisseria (23,24). Additional glycosylation sites have been found in class II strains of Neisseria meningitidis where they have been hypothesized to play a role in immune evasion (25).Among the man...

The structure of PilA from Acinetobacter baumannii AB5075 suggests a mechanism for functional specialization in Acinetobacter type IV pili

“…The final branch contains representatives from all three species, including PilA AB5075 , D. nodosus FimA from serotypes D and H, and P. aeruginosa PilA 1244 . We noted the existence of this third branch in the dendogram previously (15). However, unlike the ACICU and BIDMC57 division, we could find no ready explanation for the division between the ACICU and AB5075 branches.…”

“…Previously, we solved X-ray crystal structures of PilA, the major pilin protein, from two strains of A. baumannii: ACICU and BIDMC57 (15). PilA ACICU and PilA BIDMC57 were much more similar to pilin structures from Pseudomonas aeruginosa and Dichelobacter nodosus than to each other, seemingly products of convergent evolution.…”

“…However, a phylogenetic analysis of amino acid sequences from the major pilins of A. baumannii, P. aeruginosa, and D. nodosus showed three clusters, each containing sequences from multiple species (15). We chose to examine the structure of PilA from A. baumannii AB5075, a recently characterized and unusually virulent clinical isolate (28), both as a representative of this third taxonic group and because unlike PilA ACICU and PilA BIDMC57 , PilA AB5075 is not natively C-terminally glycosylated.…”

“…host cell adherence (15). T4P are ubiquitous within the Acintobacter genus, which contains environmental strains from soil and water, as well as commensal and pathogenic strains isolated from mammalian hosts, including humans (16 -18).…”

Bacteria defend against phages through type IV pilus glycosylation

“…A convincing reason for the prevalence of pilin glycosylation in nature has not yet been put forward, though this modification has a modest effect on pathogenicity in mice 28 and decreases twitching motility on plastic 29, 30 . In Neisseria meningitidis , an obligate human commensal, and in the multidrug resistant genus Acinetobacter , pilin glycosylation was proposed to block binding of pilin–specific antibodies 31, 32 . Here we show that modification of P. aeruginosa pilins by either of its glycosylation systems blocks infection by many pilus-specific phages.…”