Characterization of Novel appendages found on Pseudomonas aeruginosa

Abstract

The current increase in incidence of multi-drug resistant (MDR) Pseudomonas aeruginosa among critically ill patients is a worldwide concern. A recent study has shown that several MDR clinical isolates of P. aeruginosa form previously undescribed appendages on their cell surfaces, and has identified two different types of periplasmic phosphate-binding proteins, DING and PstS, as components of the appendage. However, what roles these appendages play, and how they form still remain unknown. Here we explore the possible mechanisms of appendage formation. Using anti-DING and anti-PstS antibodies in western blotting, we confirmed that the expression of DING and PstS is completely suppressed when bacteria are grown in high phosphate conditions, yet we observed that the bacteria produce appendages in these conditions, suggesting that the proteins are not the essential components of the appendages. Using a lipopolysaccharide (LPS) extraction kit, we showed that the amount of LPS extracted from bacteria that produce different numbers of appendages is similar, which rejects the hypothesis that LPS production or concentration may drive the appendage assembly. The observation that flagella-negative mutant strains produce appendages also rules out the possibility that flagella may play an important role during appendage formation. By cross-sectioning the appendages, we were able to observe the outer membrane (OM) phospholipid bilayer structure on the surface of the appendages, which led to an idea that the appendages could form by budding from the bacterial OM. This idea was further supported by the mass spectrometry data that indicates the purified appendages consist largely of OM proteins, and by the evidence that the amount of proteins, especially PstS, inside the periplasm affects the number of appendages produced. The pseudomonas quinolone signal (PQS) which is known to destabilize the bacterial OM, when added exogenously, enhanced the outer membrane vesicle formation in MDR25 strain, but not in PA14 strain. The result thus seems to indicate that two strains have OMs that are different in strength or integrity, which may partly explain the different number of appendages they produce. Overall, this study suggests that the DING/PstSrich appendages identified in the previous study are the elongated form of DING/PstScontaining OM blebs.