Herein we report that peptide dendrimers G3KL and TNS18, which were recently reported to control multidrug-resistant bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, strongly inhibit biofilm formation by P. aeruginosa PA14 below their minimum inhibitory concentration (MIC) value, under which conditions they also strongly affect swarming motility. Eradication of preformed biofilms, however, required concentrations above the MIC values. Scanning electron microscopy observation and confocal laser scanning micrographs showed that peptide dendrimers can destroy the biofilm morphological structure and thickness in a dose-dependent manner, even make the biofilm dispersed completely. Membrane potential analysis indicated that planktonic cells treated with peptide dendrimers presented an increase in fluorescence intensity, suggesting that cytoplasmic membrane could be the target of G3KL and TNS18 similarly to polymyxin B. RNA-seq analysis showed that the expressions of genes in the arnBCADTEF operon-regulating lipid A modification resulting in resistance to AMPs are differentially affected between these three compounds, suggesting that each compound targets the cell membrane but in different manner. Potent activity on planktonic cells and biofilms of P. aeruginosa suggests that peptide dendrimers G3KL and TNS18 are promising candidates of clinical development for treating infections.
FleQ plays a crucial role in motility and biofilm formation by regulating flagellar and exopolysaccharide biosynthesis in Pseudomonas aeruginosa . It has been reported that the expression of FleQ is transcriptionally downregulated by the virulence factor regulator Vfr. Herein we demonstrated that a LysR-type transcriptional regulator, OsaR, is also capable of binding to the promoter region of fleQ and repressing its transcription. Through gel shift and DNase I footprinting assays, the OsaR binding site was identified and characterized as a dual LysR-type transcriptional regulator box (AT-N 11 -AT-N 7 -A-N 11 -T). Mutation of the A-T palindromic base pairs in fleQ promoter not only reduced the binding affinity of OsaR in vitro , but also de-repressed fleQ transcription in vivo . The OsaR binding site was found to cover the Vfr binding site; knockout of osaR or vfr separately exhibited no effect on the transcriptional level of fleQ ; however, fleQ expression was repressed by overexpression of osaR or vfr . Furthermore, simultaneously deleting both osaR and vfr resulted in an upregulation of fleQ , but it could be complemented by the expression of either of the two repressors. In summary, our work revealed that OsaR and Vfr function as two transcriptional repressors of fleQ that bind to the same region of fleQ but work separately. IMPORTANCE Pseudomonas aeruginosa is a widespread human pathogen, which accounts for serious infections in the hospital, especially for lung infection in cystic fibrosis and chronic obstructive pulmonary disease patients. P. aeruginosa infection is closely associated with its motility and biofilm formation, which are both under the regulation of the important transcription factor FleQ. However, the upstream regulatory mechanisms of fleQ have not been fully elucidated. Therefore, our research identifying a novel regulator of fleQ as well as new regulatory mechanisms controlling its expression will be significant for better understanding the intricate gene regulatory mechanisms related to P. aeruginosa virulence and infection.
Bacteria have evolved distinct molecular mechanisms as a defense against oxidative stress. The foremost regulator of oxidative stress response has been found to be OxyR. However, the molecular details of regulation upstream of OxyR remain largely unknown and need further investigation. Here, we characterize a oxidant stress and antibiotic tolerance regulator, OsaR (PA0056), produced by Pseudomonas aeruginosa. Mutation of osaR increased bacterial tolerance to aminoglycoside and beta-lactam antibiotics, as well as to hydrogen peroxide. Expression of the oxyR regulon genes oxyR, katAB, and ahpBCF was increased in the osaR mutant. However, the OsaR protein does not regulate the oxyR regulon genes through direct binding to their promoters. PA0055, osaR, PA0057 and dsbM are in the same gene cluster, and we provide evidence that expression of these genes involved in oxidant tolerance is controlled by binding of OsaR to intergenic region between osaR and PA0057, which contain two divergent promoters. The gene cluster is also regulated by PA0055 via an indirect effect. We further discovered that OsaR formed intramolecular disulfide bonds when exposed to oxidative stress, resulting in a change of its DNA binding affinity. Taken together, our results indicate that OsaR is inactivated by oxidative stress and plays a role in the tolerance of P. aeruginosa to aminoglycoside and beta-lactam antibiotics. IMPORTANCE As opportunistic pathogen, Pseudomonas aeruginosa can cause serious infections which are hard to eradicate because of antibiotic resistance in immunodeficient patients. We found that OsaR is involved in oxidative stress and antibiotics resistance by regulation of downstream genes via redox state change. Research on factors affecting the transcriptional level of oxyR is very limited, but important since it has implications on antibiotic resistance. In this study, it was found that OsaR can indirectly inhibit transcription of oxyR. In addition the gene cluster composed of PA0055, osaR, PA0057 and dsbM was identified, and the associated regulatory mechanisms and functions were elucidated. Our work not only provides a mechanistic understanding of antibiotic tolerance regulation in P. aeruginosa, but also has significant implications for redox regulation in human pathogens in general.
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