Enhancing pili assembly and biofilm formation in Acinetobacter baumannii ATCC19606 using non-native acyl-homoserine lactones

Luo et al. BMC Microbiology Enhancing pili assembly and biofilm formation in Acinetobacter baumannii ATCC19606 using non-native acyl-homoserine lactones Li-mei Luo 0 Li-juan Wu 0 Yu-ling Xiao Dan Zhao Zhi-xing Chen Mei Kang Qi Zhang Yi Xie 0 Equal contributors Department of Laboratory Medicine, West China Hospital of Sichuan University , Chengdu 610041 , China Background: Quorum Sensing (QS) systems influence biofilm formation, an important virulence factor related to the bacterial survival and antibiotic resistance. In Acinetobacter baumannii, biofilm formation depends on pili biosynthesis, structures assembled via the csuA/BABCDE chaperone-usher secretion system. QS signaling molecules are hypothesized to affect pili formation; however, the mechanism behind this remains unclear. This study aimed to demonstrate the possible role of QS signaling molecules in regulating pili formation and mediating the ability to form biofilms on abiotic surfaces. Results: Real-time quantitative PCR analysis showed the expression of the csuA/BABCDE genes distinctly increased when co-cultured with C6-HSL (P < 0.05). Under the same experimental conditions, expression of BfmS and BfmR was significantly higher than the control strain (P < 0.05). A subsurface twitching assay showed a switch from a small to a large and structured clone that may result from enhanced twitching motility (P < 0.05). Transmission electron microscopy analysis of cells lifted from a MH broth co-cultured with C6-HSL showed more abundant pili-like structures than the control strain. We then tested the idea that the addition of a QS signal, and therefore induction of chaperone-usher secretion system genes, provides a greater benefit at higher biofilm densities. An assay for the total fluorescence intensity of the biofilm using Confocal Laser Scanning Microscopy revealed an obvious increase. Conclusion: Our study demonstrated that, increased transcription of the BfmS and BfmR genes, QS signaling molecules enhance the expression of the chaperone-usher secretion system, and this expression is required for twitching motility in A. baumannii. The concomitant pili expression and strain twitching allowed A. baumannii to attach easily to abiotic surfaces and form biofilms at an earlier timepoint. A; baumannii; Bacteria; Quorum sensing; Type IV pili; Biofilm; Bacterial adhesion - Background Acinetobacter baumannii is an important Gram-negative nosocomial pathogen often associated with severe nosocomial infections, including ventilator-associated pneumonia, urinary tract infections, bacteremia and septicemia, especially in patients hospitalized in intensive care units [1,2]. A. baumannii is highly resistant to several antimicrobial agents, conferred mainly by intrinsic expression of cephalosporinase and efflux pumps, and by formation of biofilms [3]. The biofilms of A. baumannii lead to a reduction in the accumulation of antibiotics in the biofilm polymeric matrix [4]. Biofilms are also associated with survival properties, virulence expression and bacterial communication [5,6]. Recent studies indicate biofilm development is related to quorum sensing. Quorum sensing is an important global regulatory system in bacteria that provides a mechanism to coordinate the behavior of individual bacteria in a population [7]. Biofilms provide a tertiary structure for bacterial communication mediated by quorum sensing pathways. A number of signaling molecules with the ability to modulate quorum sensing-dependen enzymes are known as regulators for biofilm formation [8,9]. In Gram-negative species, acyl-homoserine lactones (AHLs) are mainly employed as autoinducers used by bacteria to control biofilm formation and maintenance [10,11]. Along with many kinds of AHLs, the production of C6-HSL was previously found in A. baumannii clinical isolates [12], and most Acinetobacter strains showed very weak degradation activity against C6-HSL [13]. Pili of A. baumannii are encoded by the csuA/BABCDE chaperone-usher assembly system, which is controlled by a two-component regulatory system encoded by BfmS and BfmR. It was previously shown that BfmR is essential for stabilization of csu operon expression and the expression of csuC and csuE genes is involved in the initial surface attachment during biofilm formation [5,14]. These data suggest A. baumannii pili are a key factor in biofilm formation. Although quorum sensing and bacterial pili have been implicated in A. baumannii biofilm formation, there is very little known about the mechanism surrounding these signal molecules, csuA/BABCDE-mediated pili and biofilms in A. baumannii. In this study, an analysis of the processes of pili production and surface attachment of A. baumannii ATCC19606 was initiated, including the associated gene expression of csuA/BABCDE chaperoneusher complex and their regulating genes (BfmS/R). In addition, we present evidence for a possible role of quorum sensing signaling molecules in the formation of biofilms on abiotic surfaces. Results and discussion Impact of C6-HSL on chaperone-usher complex expression The capacity of A. baumannii to form biofilms is a decisive advantage for its survival in the hospital environmental. Recent studies have linked biofilm development with quorum-sensing pathways and bacterial factors, such as A. baumannii pili [15,16]. It is known that disruption of the csuC and csuE ORFs, which belong to the csuA/BABCDE Figure 1 Transcript levels of genes within the csu operon. Quantitative RT-PCR assays of ATCC19606 cells grown in LB broth without AHLs (control) or with the addition of 100 mol/L AHLs (C6-HSL). Transcription of each gene of the chaperone-usher complex were increased >1.5-fold. Figure 2 Transcript levels of the csuA/BABCDE chaperone-usher complex regulating genes BfmS/R. Quantitative RT-PCR assays of ATCC19606 cells grown in LB broth without AHLs (control) or with the addition of 100 mol/L AHLs (C6-HSL). Both genes were increased approx 1.33-fold. bacterial pili structure gene cluster, results in non-piliated cells and abolishes cell attachment [14]. However, the exact mechanism of how QS pathways and csu influence biofilm formation is unclear. To directly examine all the genetic components of the csuA/BABCDE, and their regulators, the BfmS-BfmR regulating system that includes response factor (BfmR) and sensor kinase (BfmS), we provide data on the comprehensive expression of the pili structure gene cluster and the impact of C6-HSL on this chaperone-usher secretion system. Our results showed expression of bacterial pili structure genes, including csuA/B, csuA, csuB, csuC, csuD and csuE, significantly increased after addition of 100 mol/L C6-HSL, and the transcript levels of the csuA/BABCDE chaperone-usher complex were increased >1.5-fold over the control group (P < 0.05, Figure 1). Furthermore, at the same experimental conditions, expression of chaperone-usher regulators (BfmS and BfmR) were higher than those of the control strain, and th (...truncated)