Targeting the Holy Triangle of Quorum Sensing, Biofilm Formation, and Antibiotic Resistance in Pathogenic Bacteria

Abstract

Chronic and recurrent bacterial infections are frequently associated with the formation of biofilms on biotic or abiotic materials that are composed of mono- or multi-species cultures of bacteria/fungi embedded in an extracellular matrix produced by the microorganisms. Biofilm formation is, among others, regulated by quorum sensing (QS) which is an interbacterial communication system usually composed of two-component systems (TCSs) of secreted autoinducer compounds that activate signal transduction pathways through interaction with their respective receptors. Embedded in the biofilms, the bacteria are protected from environmental stress stimuli, and they often show reduced responses to antibiotics, making it difficult to eradicate the bacterial infection. Besides reduced penetration of antibiotics through the intricate structure of the biofilms, the sessile biofilm-embedded bacteria show reduced metabolic activity making them intrinsically less sensitive to antibiotics. Moreover, they frequently express elevated levels of efflux pumps that extrude antibiotics, thereby reducing their intracellular levels. Some efflux pumps are involved in the secretion of QS compounds and biofilm-related materials, besides being important for removing toxic substances from the bacteria. Some efflux pump inhibitors (EPIs) have been shown to both prevent biofilm formation and sensitize the bacteria to antibiotics, suggesting a relationship between these processes. Additionally, QS inhibitors or quenchers may affect antibiotic susceptibility. Thus, targeting elements that regulate QS and biofilm formation might be a promising approach to combat antibiotic-resistant biofilm-related bacterial infections.

Keywords: ESKAPE bacteria; antibiotic resistance; antibiotic sensitization; biofilm; biofilm inhibitors; efflux pump inhibitors; quorum sensing; quorum sensing inhibitors.

Figure 1

(A). Induction of vancomycin resistance in Staphylococcus aureus by vancomycin. Vancomycin activates the TCS VanRS, which induces the expression of the vanHAX operon responsible for the synthesis of the D-alanyl-D-lactate dipeptide. The D-alanyl–D-lactate dipeptide shows a 1000-fold lower affinity to vancomycin compared to the regular D-alanyl–D-alanine dipeptide, thereby conferring vancomycin resistance. (B). Examples of regulatory mechanisms involved in antibiotic resistance and biofilm formation in Staphylococcus aureus. The expressions of the efflux pumps NorA and AbcA, which confer antibiotic resistance to fluoroquinolones and β-lactams, respectively, are induced by their respective substrates norfloxacin and ampicillin. Additionally, their expression levels are influenced by the ArlRS and Agr TCSs, which both affect the global transcriptional regulator MgrA (also known as NorR). Phosphorylation of MgrA by the PknB serine/theonine kinase leads to increased transcription of norA. The ArlRS and Agr TCSs are regulated by several transcriptional factors (e.g., NorG, Rot, SarA, SarR, SarZ, SigB) as illustrated in the figure. This network of regulatory factors also affects biofilm formation, where AbcA promotes biofilm formation by exporting phenol-soluble modulins (PSMs), and the Agr QS prevents biofilm formation. The Agr QS is explained in more detail in Section 3.3.

Figure 2

Induction of antibiotic resistance in Pseudomonas aeruginosa by antibiotics, zinc ions, and low concentrations of extracellular magnesium ions. The expression of the MexXY-OprM efflux pump responsible for multidrug resistance is regulated by several factors including ribosome-targeting antibiotics and the TCSs AmgRS and ParRS. AmgRS is activated by envelope stress, and ParRS is activated by the cationic antimicrobial peptide colistin as well as zinc ions. Moreover, colistin activates other TCSs including CprRS and PmrAB. The latter TCS is also affected by the TCS PhoPQ, which is activated by low extracellular magnesium ion concentrations, and extracellular DNA which sequesters magnesium ions. Extracellular DNA is a central component of Pseudomonas aeruginosa biofilms. The TCSs ParRS, CprRS, and PmrAB induce the translation of the arnBCADTEF operon which is responsible for the L-Ara4N modification of LPS, resulting in resistance to colistin. Additionally, LPS is modified by palmitoylation through PhoPQ-mediated upregulation of pagP, and by PEtN attachments regulated by the ColRS TCS.

Figure 3

Examples of antibiotic resistance mechanisms induced by bile acids. Bile salts which are secreted into the duodenum, induce the expression of various genes in bacteria that confers antibiotic resistance. Outstanding is the upregulation of efflux pumps such as MexAB-OprM in Pseudomonas aeruginosa, AcrAB-TolC in Enterobacteriaceae and EmrB/QacA in Enterococcus faecalis that confer resistance to multiple antibiotics and antiseptics, as well as to the bile salts themselves. Additionally, the TCSs BasRS and PmrAB are induced in Escherichia coli, resulting in the upregulation of the arnBCADTEF operon responsible for the L-Ara4N modification of LPS. This modification reduces the affinity of colistin/polymyxin B to LPS, with consequent resistance to these drugs.

Figure 4

Induction of antibiotic resistance by the tryptophan metabolite indole. Indole, which is produced by various gut bacteria including Escherichia coli, induces the expression of various efflux pumps (e.g., AcdD, MdtABC, AcdE, AcrAB-TolC) through activation of TCSs (e.g., BaeSR, CpxAR) or transcriptional regulators (GadX, RamA). The activity of RamA is negatively regulated by RamR.

Figure 5

Regulation of AcrAB-TolC efflux pump expression in Gram-negative bacteria. The expression of AcrAB-TolC, which confers multidrug resistance, is regulated by several transcriptional regulators including MarA, RarA, RamA, Rob, AcrR, SidA, and SoxS. MarA, in turn, is regulated by the TCSs CpxAR and QseBC, as well as various antibiotics. MarA reduces the expression of the OmpF porin which is required for the penetration of several antibiotics into the bacteria. RarA is activated by the antibiotic ertapenem. Besides upregulating AcrAB-TolC, RarA increases the expression of the OqxAB multidrug efflux pump. Bile acids and fatty acids increase AcrAB-TolC expression through repression of the Rob transcriptional regulator. SoxS is regulated by SoxR whose activity is influenced by oxidative stress, as well as by the Pseudomonas aeruginosa-produced pyocyanin pigment. In turn, SoxS increases the expression of SodA superoxide dismutase, which is a mechanism to protect the bacteria from oxidative stress. Additionally, SoxR induces the expression of the MexHI-OpmD efflux pump, thus conferring resistance to additional compounds.

Figure 6

Regulation of MexAB-OprM multidrug efflux pump expression in Pseudomonas aeruginosa. The expression of the MexAB-OprM efflux pump is positively and negatively regulated by a whole range of transcriptional regulators. Its expression is also affected by bile salts and induced by the AmgRS TCS and CpxR, which is the cognate response regulator of the CpxAR TCS.

Figure 7

Regulation of the antiholin LrgA–holin CidA system by the small SprX RNA. SprX inhibits the expression of the RNA-binding protein SpoVG, which positively regulates the TCS LytSR. LytSR positively regulates the antiholin LrgA which antagonizes the activity of the holin CidA. SpoVG induces methicillin and oxacillin resistance by promoting cell wall synthesis and inhibiting cell wall degradation. LrgA and CidA affect the response to penicillin by respectively inhibiting or activating murein hydrolase activities. Altogether, the overexpression of SprX results in increased susceptibility to β-lactams.

Figure 8

Crosstalk of the four QS systems Las, Rhl, Pqs, and Iqs in Pseudomonas aeruginosa and their relationship to biofilm formation. Each of the four TCS systems produces its own autoinducer (C12-HSL, PQS, IqsR, or C4-HSL) that acts on the corresponding receptor (LasR, PqsR, IqsR, or RhlR). The receptors, in turn, elicit phosphorelay cascades resulting in altered expression of a large number of genes. In addition, there is crosstalk between these TCSs. LasR affects PqsR, IqsR, and RhlR. There is also mutual communication between PqsR and RhlR. LasR is involved in biofilm development and maturation. PqsR is involved in early biofilm formation and the release of extracellular DNA. RhlR is involved in the maintenance of biofilm channels and the detachment of biofilm-embedded cells. IqsR promotes biofilm formation by acting on PqsR and RhlR.

Figure 9

The Agr QS system of Staphylococcus aureus. The arg operon consists of the 4 genes: agrB, agrD, agrC, and agrA. AgrD encodes for a precursor of the autoinducer peptide AIP that is processed and transported through the cell membrane by AgrB. The mature AIP interacts with its receptor AgrC eliciting a phosphorelay, resulting in the phosphorylation and activation of the response regulator AgrA that affects the expression of multiple genes including the genes of the agr operon by binding to the P2 promoter, and the RNAIII regulatory RNA by binding to the P3 promoter. RNAIII affects the expression of a large number of genes, thereby promoting the virulence of Staphylococcus aureus. A small part of the RNAIII transcript encodes for the δ-toxin (delta-hemolysin; hld). Additionally, AgrA activates the TCSs ArlRS and SaePQRS, as well as upregulating the expression of the virulence factors phenol-soluble modulins (PSMs) alpha and beta. As shown in Figure 1B, ArlRS modulates the expression of the NorA efflux channel responsible for fluoroquinolone resistance and regulates virulence factors through the induction of MgrA. The SaePQRS TCS regulates the expression of various virulence factors.

Figure 10

The Cpx envelope stress-response system. The CpxAR TCS is activated by various stress stimuli including protein misfolding, certain antibiotics, inner membrane disruption, alkaline pH, starvation, high osmolarity, and adherence to an abiotic surface. The activation of CpxAR leads to large alterations in gene expression in an attempt to protect the bacteria from the environmental stressor. Among others, it induces the expression of genes assisting in removing misfolded proteins and genes involved in biofilm formation. Additionally, CpxAR increases the expression of various genes (e.g., efflux pumps, porins, amidases, and the ldtD transpeptidase) that confer multidrug resistance and copper tolerance.

Figure 11

The role of c-di-GMP in biofilm formation and antibiotic resistance. The secondary metabolite c-di-GMP is a central mediator of biofilm formation in Gram-negative bacteria. It modulates the expression of various genes (e.g., fimX, alg44, pelD, fleQ) involved in the production of pili, flagella, alginate, and exopolysaccharides (Pel and Psl) that contribute to the development of the biofilm. Moreover, it induces the transcriptional responser BrlR that regulates the expression of efflux pumps involved in antibiotic resistance. The activity of BrlR is affected by SagS, which is involved in the molecular switch from a planktonic to a biofilm lifestyle (see Figure 12 below). The c-di-GMP level is affected by various factors including cell adhesion and cell envelope stress. Its synthesis is mediated by diguanylate cyclases (DGCs) and degraded by c-di-GMP phosphodiesterases (PDEs).

Figure 12

(A). Regulation of RsmY/RsmZ small RNAs by the GacSA TCS and c-di-GMP in Pseudomonas aeruginosa and the impact on biofilm formation. RsmY and RsmZ are small regulatory RNAs involved in the regulation of biofilm formation. Their expression is regulated by the TCS GacSA, whose activity is, in turn, regulated by the LadS, RetS, and P1611 kinases. RsmY and RsmZ sequester the translational repressor RsmA, thereby relieving its inhibitory action on c-di-GMP production and biofilm formation. Since RsmA increases motility through the type III secretion system (T3SS), an increase in the RsmY and RsmZ levels would result in reduced motility. (B). Additional TCS components regulating biofilm formation. In the planktonic state, SagS is phosphorylated and prevents the activity of BfiSR. Dephosphorylation of SagS leads to phosphorylation of BfiSR resulting in the switch from a reversible attachment to an irreversible attachment. This is followed by phosphorylation of BfmSR, which initiates biofilm maturation, and phosphorylation of MifSR, which is required for full biofilm maturation.