Host Receptor Pili for Cryo-EM Single-Particle Reconstruction

Background

Acinetobacter, a Gram-negative bacterium, is an aerobic, non-flagellated, and widely distributed coccobacillus [2]. Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter pittii, and Acinetobacter nosocomialis are opportunistic pathogens known for causing hospital-acquired infections. Bacteria can invade tissue and evade immune response through various mechanisms, including the production of enzymes. Adherence to the host is facilitated by structures such as pili [3–5]. Each year, U.S. hospitals report around 12,000 infections caused by multidrug-resistant Acinetobacter species. The protein secretion processes in Acinetobacter, which are critical targets for vaccine development, are not well understood. Known protein secretion systems in Acinetobacter include the Type II secretion system (T2SS), Type VI secretion system (T6SS), Type IV pilus secretion system, autotransporters, and outer membrane vesicles (OMVs) [6].

In this protocol, we focus on the type IV pili purification from Acinetobacter genomosp. 16 (A. gp16, NCBI: txid70347), which is a target host receptor of single-stranded RNA phage AP205 (ssRNA phage AP205, a virus that can infect Acinetobacter) [7]. Given the crucial role retractile pili play in the virulence of many bacteria, if infection-dependent pili detachment is a common feature of ssRNA phages, these simple viruses could potentially be used to inhibit retractile pili deployment and thereby decrease the virulence of numerous significant bacterial infections [7]. The purified pili can not only be utilized for Cryo-EM structural studies but also for biochemical or biophysical experiments to test protein binding affinity.

Type IV pili are extracellular helical appendages, typically with a diameter of 6–9 nm [8], which are composed of thousands of noncovalently linked pilin subunits. The major pilin of Acinetobacter type IV pili is the PilA monomer, which is composed of an N-termini hydrophobic α-helix, an α-β loop for connection, and the C-termini soluble β-sheet. The mature pilins assemble into type IV pilus by type IV pilus secretion system. Pilins bury the N-terminus helix inside, forming the pilus’s central axis, with the C-terminus headgroup (β-sheet) exposed outside [9].

Several protocols have been established for the purification of different types of pili [1,10,11]. While these methods share commonalities, specific steps can vary depending on the bacterial strain. We tested various pili purification techniques and found that the method developed by Costa et al. [1] for Escherichia coli (E. coli) F-pili provided higher efficiency and yield than other methods. First, we optimized and highlighted critical steps within this protocol to improve and stabilize yield, such as using a syringe to facilitate pili detachment from cells and controlling temperature to prevent pili retraction. Second, we focused on optimizing the procedure for Acinetobacter pili purification, allowing for the collection and separation of two different types of pili simultaneously. There are two types of pili outside the cell boundary: type IV pili and FilA filament. Lastly, this paper outlines a four-section process and provides two options (one for hyper-piliated strains, the other for low-yield strains), making it suitable for pili from other bacteria species as well (Graphical overview). We found that some steps could be simplified when bacterial pili yield is high. For instance, Acinetobacter pili have a relatively higher yield compared to F-pili, which have only one or two pili per cell. Microscope negative staining can be used to visually assess the number of pili per cell, determining whether the strain is hyper-piliated or has a lower yield. Based on this assessment, one can decide whether to follow the method in Sections B and C or Sections B2 and C2. Sections B and C provide a quicker method for obtaining pili from Acinetobacter and other high-yield species. Additionally, alternative steps in Sections B2 and C2, which mainly optimize the method from Costa et al. [1] are provided for strains with lower pili yields (e.g., E. coli F-pili).

In summary, this protocol has been optimized based on previous pili purification methods to enhance yield and stability, particularly for Acinetobacter pili. This method can also be generalized for the purification of pili in other species.