Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen affecting immunocompromised patients. It is known as the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and as one of the leading causes of nosocomial infections. Due to a range of mechanisms for adaptation, survival and resistance to multiple classes of antibiotics, infections by P. aeruginosa strains can be life-threatening and it is emerging worldwide as public health threat. This review highlights the diversity of mechanisms by which P. aeruginosa promotes its survival and persistence in various environments and particularly at different stages of pathogenesis. We will review the importance and complexity of regulatory networks and genotypic-phenotypic variations known as adaptive radiation by which P. aeruginosa adjusts physiological processes for adaptation and survival in response to environmental cues and stresses. Accordingly, we will review the central regulatory role of quorum sensing and signaling systems by nucleotide-based second messengers resulting in different lifestyles of P. aeruginosa. Furthermore, various regulatory proteins will be discussed which form a plethora of controlling systems acting at transcriptional level for timely expression of genes enabling rapid responses to external stimuli and unfavorable conditions. Antibiotic resistance is a natural trait for P. aeruginosa and multiple mechanisms underlying different forms of antibiotic resistance will be discussed here. The importance of each mechanism in conferring resistance to various antipseudomonal antibiotics and their prevalence in clinical strains will be described. The underlying principles for acquiring resistance leading pan-drug resistant strains will be summarized. A future outlook emphasizes the need for collaborative international multidisciplinary efforts to translate current knowledge into strategies to prevent and treat P. aeruginosa infections while reducing the rate of antibiotic resistance and avoiding the spreading of resistant strains.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; biofilm; persistence; virulence.

Figure 1

Hierarchical QS network in P. aeruginosa and regulation of virulence factors. (A) So far, four pathways including Las, Rhl, Pqs, and IQS have been understood as mediating QS responses in P. aeruginosa while LasR resides at the top of the cascade. In response to specific stimuli/stress, each pathway synthesizes cognate autoinducers (AIs) [HSL(3-oxo-C12-homoserine lactone)], BHL (N-butyrylhomoserine lactone or C4-HSL), PQS (2-heptyl-3-hydroxy-4-quinolone) and IQS [2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (aeruginaldehyde)]. Export and import of HSL, BHL, and PQS is mediated by the efflux pumps MexAB-OprM/MexEF-OprN, free diffusion and membrane vesicles, respectively. Question mark indicates unknown pathway of IQS transportation. As fine-tuned individual circuits, but interconnected (dashed lines), transcriptional factors (i.e., LasR, RhlR, and PqsR) are activated by AIs for upregulating expression of cognate AI synthases (respectively, LasI, RhlI, PqsABCDH) as well as others such as virulence factor genes. The IQS pathway remains unraveled and the IQS receptor is still unknown. Various secretion systems mainly type 1 and 2 secretion systems (T1SS/T2SS) and also PvdRT-OpmQ efflux pump mediate the secretion of virulence factors. (B) QS initiates upon cumulative production of AIs (small colorful circles) by increasing cell density and results in collective responses. AprA, alkaline protease; Pyd, pyoverdine; PLC, phospholipase C; Tox, toxin A; LasA, LasA elastase; LasB, LasB elastase; HCN, hydrogen cyanide; Pyo, pyocyanin; Rhld, rhamnolipids; Lec A, lectin A; CM, cytoplasmic membrane; OM, outer membrane.

Figure 2

Regulatory networks underlying biofilm formation by P. aeruginosa. (A) Elevation of the cyclic di-GMP molecule is a key determinant for the motility-sessility switch. Environmental cues are sensed by various proteins localized in the envelope of the cells where these proteins contribute to two-component systems (brown/green rectangles), chemoreceptor-like system (orange complex) and other receptor mediated signaling pathways (arranged in the left side of figure). Either triggered as phosphorylation cascades (small red circle) or protein-protein interactions, the signals induce diguanylate cyclases (containing GGDEF motif) (red rectangles) to synthesize cyclic di-GMP from two molecules of GTP (guanosine-5′-triphosphate). Consequently, cyclic di-GMP sensing proteins act as receptor/effector for specific outputs such as induction of alginate and Pel polymerization, inhibition of motility and derepression of psl/pel expression via FleQ, induction of attachment and biofilm formation/maturation triggered by two component systems. The two-component systems are interconnected and the LadS/RetS/GacS/GacA/RsmA regulatory network (green rectangles) plays a key role in the phenotypic switch from motility to sessility and downregulation of QS and virulence factor production. (B) Various stages of biofilm formation and development were represented. Plus and minus signs represent positive and negative effect of transcriptional regulators, respectively. CM, cytoplasmic membrane; OM, outer membrane.

Figure 3

Intrinsic, acquired and adaptive mechanisms confer antibiotic resistance in P. aeruginosa. For each mechanism, various molecular strategies, which confer resistance to specific class of antipseudomonal antibiotics (Car., Carbapenems; Ceph., Cephalosporins; Pen., Penicillins; Ami., Aminoglycosides; Flu., Fluoroquinolones; Mac., Macrolides and Pol., Polymyxins), were presented at the top of the figure (underlined) Intrinsic mechanisms such as structural barriers [e.g., EPS (extracellular polymeric substances)], OprD reduction and basal production of AmpC β-lactamase and MexAB/XY efflux pumps confer a basal resistance to some group of antibiotics. However, in acquired resistance, mutational changes in the oprD gene, transcriptional repressors causingupregulation of resistance genes and efflux pumps conferring resistance against a wider spectrum of antibiotics. Plasmid-mediated resistance is very potent as a variety of resistance genes can be exchanged among bacteria. Either mediated by mutational changes in the genome or in plasmids, resistance to polymyxins occurs via modification of LPS (lipopolysaccharide) components hindering binding of the antibiotic to this layer. Adaptive resistance occurs in the presence of antibiotics mainly via mutation in regulatory genes. This is a transient and reversible resistance, which will reverse upon removal of antibiotics. Stars represent antibiotics and dashed/wavy lines represent transcriptional levels of each gene product. CM, cytoplasmic membrane; OM, outer membrane.

Figure 4

Remodeling of regulatory networks in P. aeruginosa during adaptive radiation and transition from acute to chronic infections. (A) During pathogenesis, adaptation to the CF lung environment occurs through “adaptive radiation” where intense genetic mutations lead to diverse genotypes and phenotypes (colorful ellipsoids) within bacterial populations followed by the selection of colonizers. Mutational adaptation and selection of generations drive bacterial transition from acute to chronic traits. (B) Remodeling of key regulatory networks between acute and chronic infections occurs mainly via mutational adaptation in cognate genes. Mutated lasR, ampR, and retS genes are key determinants in this process by which QS, virulence factor production and motility are downregulated, while synthesis of cyclic di-GMP, exopolysaccharides and various multidrug efflux pumps are upregulated. Mutation of mucA results in a defect in MucA (anti-sigma factor) releasing AlgU (positive regulator of alginate operon) that induces overproduction of alginate and the mucoid phenotype. Of important acute traits are flagellum, type 4 pili, T1SS, T2SS, T3SS (types 1 to 3 secretion systems), ExoT (exotoxins), Lip (lipases), AprA (alkaline proteases). The type 6 secretion system (biofilm-associated and toxin-delivering device to other bacteria) and efflux pumps and the production of EPS are part of chronic traits which confer antibiotic resistance and/or mediate biofilm formation. Plus and minus signs represent positive and negative effect of transcriptional regulators, respectively. Red cross indicates mutagenesis. CM, cytoplasmic membrane; OM, outer membrane.

Figure 5

P. aeruginosa stringent response and persister formation. Stringent response is triggered by particular stresses such as amino acid and fatty acid starvation, iron/phosphor depletion and oxidative stress [e.g., reactive oxygen species (ROS)]. The (p)ppGpp alarmone is a key determinant for stringent response and it is elevated by RelA/SpoT enzymes. Generally, (p)ppGpp elevation and the PolyP (inorganic polyphosphate) and Lon protease complex interfere with normal biological processes in favor of bacterial survival via arresting metabolism, cell growth and cell division (dashed gray pathways are best understood for the E. coli model, but not or partially characterized in P. aeruginosa). In E.coli, (p)ppGpp signaling is linked to toxin (T)-antitoxin (A) system via activation of the Lon protease leading to the formation of persisters displaying dormant and antibiotic resistance phenotypes (dashed orange line). Generally, the TA complex is stable under normal conditions suppressing toxin activity and further expression of cognate genes. Upon antitoxin degradation, toxin becomes active to hinder biological processes. In the case of P. aeruginosa HigB/A, HicA/B, the toxin components perform endoribonuclease (RNase) activity on mRNA molecules. In P. aeruginosa, the (p)ppGpp alarmone is linked to the production of ROS scavengers probably via QS or RpoS regulators and Lon activity is required for biofilm formation, motility, virulence and antibiotic resistance. Furthermore, the TA system downregulates biofilm formation and virulence factor production while T3SS (type 3 secretion system) can be found upregulated. Although, the (p)ppGpp signaling, Lon protease activity and TA modules (i.e., HigB/A, HicA/B, and likely more complexes) are present in P. aeruginosa, their link to resistance to antibiotics and other stresses is poorly understood. AA, amino acids; QS, quorum sensing; RNAP, RNA polymerase. CM, cytoplasmic membrane; OM, outer membrane.