Background

Pseudomonas aeruginosa is an opportunistic pathogen that often causes serious hospital-acquired infections and can lead to a variety of infectious diseases including pneumonia, wound infections, and septicemia [1]. Due to the high mortality rate of these infections, P. aeruginosa was recognized as one of the most life-threatening bacteria and listed as a priority pathogen for research and development of new antibiotics by the World Health Organization [2].

The Pf phage, a filamentous inovirus specifically associated with P. aeruginosa, is a type of temperate phage, which can be classified from Pf1 to Pf8. Approximately half of P. aeruginosa isolates harbor Pf phage operons within their genomes [3,4]. Pf phages are about 6–7 nm in diameter and vary in length from 0.8 to 2 µm, with a single-stranded DNA (ssDNA) as the genetic material packaged within a helical filamentous structure major coat protein CoaB [3]. Of these, Pf4 phage is an important member, and its genome is integrated into the P. aeruginosa PAO1 as the prophage. Thus, Pf4 phage can emerge inside the bacteria [5]. The core genes of the Pf4 phages mainly include xisF4, PA0720, PA0723, PA0724, and PA0726, which encode excisionase, single-stranded DNA binding protein, major coat protein CoaB, minor coat protein CoaA, and morphogenesis protein, respectively [3,5]. A recent study showed that Pf4 phages can be transmitted between bacteria and have various effects on P. aeruginosa, including reducing twitching motility, altering biofilm production, and affecting the release of virulence factors, which are important in the pathogenesis of P. aeruginosa [6–8]. However, how Pf4 is transmitted has remained elusive.

Bacterial membrane vesicles (MVs) are structures produced by bacteria with a diameter ranging from 40 to 400 nm that carry specific cargo and travel a long distance (centimeter scale) to play essential roles in cell–cell communications [9,10]. Gram-negative bacteria MVs could be divided into two types: B (blebbing)-type vesicles, which are formed via blebbing from the outer membrane, and E (explosive)-type vesicles, which are formed due to explosive cell lysis [11,12]. Studies have suggested that P. aeruginosa E-MVs can be formed under certain stresses and bacteriophage lysis conditions induced by Pf4 phages [3].

Many previous works observed that after the crude MV extraction step, flagella can also be observed by TEM [13]. A recent study investigated the combined effects of P. aeruginosa MVs with phages upon the recognition function of the host’s innate immune system. In a mouse model of acute lung infection, treatment with MVs-Pf4 phages complex resulted in reduced infiltration of neutrophils in the lung alveoli, thus suppressing the occurrence of inflammatory reactions [3].

In this protocol, we establish a bacterial MVs–phages co-extraction platform from P. aeruginosa PAO1 as a model organism. The MVs–phages extract effectively mimics the complex MVs–phage coexistence condition in vivo within the host during pathogen infections. Overall, this is an optimized method for extracting the MV–phage complex based on density gradient centrifugation (Figure 1), with feasibility verified through TEM, PCR, and plaque assays. It will provide a reliable protocol for studying bacteriophage–microbe–host cell interactions.

Figure 1. Flowchart of MV–phage complex extraction. The bacterial culture is centrifuged to retain the supernatant. After filtering and ultrafiltering, bacteria and small molecules can be removed. After density gradient ultracentrifuging, flagella and pili can be removed. Then, purified MVs–phage samples can be obtained.