Small Molecules That Sabotage Bacterial Virulence

Abstract

The continued rise of antibiotic-resistant bacterial infections has motivated alternative strategies for target discovery and treatment of infections. Antivirulence therapies function through inhibition of in vivo required virulence factors to disarm the pathogen instead of directly targeting viability or growth. This approach to treating bacteria-mediated diseases may have advantages over traditional antibiotics because it targets factors specific for pathogenesis, potentially reducing selection for resistance and limiting collateral damage to the resident microbiota. This review examines vulnerable molecular mechanisms used by bacteria to cause disease and the antivirulence compounds that sabotage these virulence pathways. By expanding the study of antimicrobial targets beyond those that are essential for growth, antivirulence strategies offer new and innovative opportunities to combat infectious diseases.

Keywords: antivirulence therapies; bacterial pathogenesis.

Figure 1. Two-component regulatory sensor transduction systems

A prototypical two-component sensor system (TCS) is composed of a histidine kinase (HK) and a response regulator (RR). Upon sensing the environmental signal, the HK undergoes autophosphorylation at a conserved histidine residue. The phosphate is transferred to the response regulator, which typically dimerizes and acts as a transcription factor to alter expression of virulence genes. All inhibitors are shown in red and associated steps at which they function to inhibit TCS signaling. Ethoxzolamide inhibits carbonic anhydrase activity in Mycobacterium tuberculosis, leading to downregulation of the virulence associated PhoPR regulon. LED209 directly binds to conserved lysine residues on the HK QseC and inhibits activation of virulence genes in multiple pathogens. Artemisinin targets heme carried by the Mtb DosS and DosT HK to inactivate the kinases. HC102A and HC103A inhibit DosS HK autophosphorylation and HC103A also inhibits DosT autophosphorylation. A collection of NSC inhibitors inhibit the formation of the Salmonella enterica PhoP-DNA complex in vitro. This model is derived and modified from Rasko et al. [39]

Figure 2. Chemical structures of antivirulence compounds discussed in this review

Small molecules that inhibit: A. Two-component regulatory systems; B. Bacterial adherence; C. Toxins and secretion systems; and D. Metabolic pathways required for virulence.

Figure 3. Bacterial adherence mechanisms through Type I, P pili, and curli biogenesis

A. Graphical representation of type I and P pili biogenesis in Gram-negative pathogens, such as uropathogenic E. coli (UPEC). Unfolded pilin subunits are translocated through the SecYEG translocon into the periplasm. A chaperone (FimC/PapD) folds and stabilizes the subunit, passing it to the secretion machinery for incorporation into the growing appendage. FimD/PapC receives the folded subunit from the chaperone and adds the given subunit to the actively polymerizing pilus. Biaryl mannoside 22 inhibits the mannose binding capacity of the terminal subunit (FimH/PapG) in type I and P pili. Nitazoxanide inhibits pore formation of the secretion/polymerization complex (FimD/PapC). Biaryl 2-pyridone 2c binds to the surface of the chaperone (FimC/PapD) and prevents the subunit transfer to the secretion/polymerization complex (FimD/PapC). B. Graphical representation of curli biogenesis in UPEC. Unfolded curli subunits (CsgA/CsgB) are translocated into the periplasm by the SecYEG secretion machinery. CsgC is proposed to act as a chaperone, preventing premature amyloid formation by the unfolded curli subunits [186]. CsgE and CsgF act as soluble chaperones to stabilize CsgA/CsgB and transfer the unfolded subunits to the outer membrane assembly protein CsgG. CsgB acts as a nucleation factor for CsgA amyloid formation and deposition onto the growing appendage. FN075 inhibits CsgA polymerization and due to its parent structure, also acts as an inhibitor similar to the biaryl 2-pyridone 2c. All inhibitors are denoted in red. OM – bacterial outer membrane; IM – bacterial inner membrane. This model is derived and modified from Costa et al. [85]

Figure 4. Proposed mechanisms for toxtazins A and B inhibition of cholera toxin and toxin co-regulated pilus production

ToxT regulates transcription of genes responsible for producing cholera toxin (CT) and the toxin co-regulated pilus (TCP), which are two virulence factors involved in cholera infections. Toxtazin A inhibits toxT transcription and is proposed to act by causing a general stress response, feeding back to shut down transcription of toxT. Toxtazin B inhibits transcription of tcpPH and is proposed to act by reducing intracellular levels of TcpP, leading to reduced transcription of toxT. Inhibitors are in red. OM – bacterial outermembrane; IM – bacterial inner membrane. This model is derived and modified from Anthouard et al. [26]

Figure 5. Targeting C. difficile oxin processing in host cells

TcdA and TcdB are the two toxins produced by disease-associated C. difficile. Host processing of the toxins are mediated by endocytosis of the full-length toxin into an acidified compartment, leading to surface exposure of the cysteine protease domain (CPD) and glucosyltransferase domain (GTD). Interaction of the CPD with 1D-myo-inositol hexakisphosphate (IP₆) leads to activation and autocatalysis of the linker region between the CPD and GTD. GTD is released into the host cytoplasm and alters intracellular signaling through glucosylation of Rho/Rac GTPase activity. Ebselen inhibits the protease activity of CPD, preventing the release of GTD into the host cytoplasm. The Ebselen inhibitor is in red. This model is derived and modified from Bender et al. [132]

Figure 6. Type III secretion system for delivery of bacterial effectors directly into the host cytoplasm

The type III secretion system (T3SS) resembles a syringe-like structure that bacterial pathogens use to inject effectors directly into the host cytoplasm. The terminal tip of the T3SS interacts with the host cell, creating a pore for active (ATP-dependent) delivery of substrates that disrupt host signaling, lead to cytoskeletal rearrangements, and inflammation as a few examples. Yersina pestis uses effector proteins known as Yersinia outer proteins (Yops) to cause disease. Within the bacterial cytoplasm the chaperone known as specific Yersinia chaperone (Syc) stabilizes and partially folds Yops for delivery to the T3SS. The ATPase YscN is responsible for dissociating the chaperone-effector complex and failure to do so results in bacterial attenuation. The compounds 7086, 7812, and 7832 inhibit YscN ATPase activity in vitro. The HK QseC is a global virulence regulator in several Gram-negative pathogens including S. Typhimurium. QseC has been shown to globally regulate the pathogenicity island associated with T3SS production and the effector sifA [34]. LED209 is a potent regulator of QseC and attenuates S. Typhimurium during infection through inhibiting virulence gene induction, including the T3SS. Additionally, the S. Typhimurium PhoPQ two-component regulatory system is implicated in regulating several virulence-associated genes including the T3SS secreted effector SrfJ [187]. The NSC inhibitors have been shown to inhibit the PhoP-DNA complex formation in vitro, potentially disrupting virulence gene expression, including srfJ, in S. Typhimurium. Inhibitors are shown in red. HM – host membrane; OM – bacterial outer membrane; IM – bacterial inner membrane; HK – histidine kinase; RR – response regulator. This model is derived and modified from Rasko et al., and Costa et al.[10, 85]

Figure 7. Mycobacterium tuberculosis type VII secretion of EsxA/EsxB

Mycobacterium tuberculosis (Mtb) possesses a type VII secretion system (T7SS) (ESX-1) for export of host effector proteins EsxA and EsxB. The current working mechanism is that T7SS substrates are targeted for secretion by an unstructured C-terminal signal sequence (similar to type IV secretion systems). The substrates EsxA and EsxB form a dimer for secretion and stabilized by the soluble chaperone EspG and potentially exported in an ATP-dependent manner. Further, the protease MycP is required for secretion, though the exact role it plays in secretion is unclear. Regulation of ESX-1 secretion is accomplished by several cellular systems including the two-component regulator MprAB through the espACD operon. BPT15 inhibits the kinase activity of the sensor kinase MprB, leading to reduced secretion of EsxA, attenuating Mtb-mediated phagosome maturation arrest. BBH7 inhibits an unknown factor leading to reduced general secretion of Mtb and survival in fibroblasts. Inhibitors are in red. MM – mycobacterial membrane; PM – plasma membrane. This model is derived and modified from Rasko et al., and Houben et al. [10] [114]

Figure 8. Inhibiting cholesterol metabolism in Mycobacterium tuberculosis

During the course of infection, Mycobacterium tuberculosis (Mtb) requires cholesterol as a carbon source for persistence and growth. Remarkably, Mtb is capable of fully catabolizing cholesterol through the proposed mechanism above. A/B ring catabolism is accomplished through a series of enzymatic and non-enzymatic steps. HsaAB likely catalyzes the hydroxylation of 3-HAS to 3-HSBNC and is inhibited by V-13-0110503 and V-13-012725. Propionyl-CoA is produced during cholesterol catabolism and is detoxified and assimilated into the TCA cycle via the methyl-citrate cycle (MCC). The small molecule V-13-009920 inhibits the first enzyme, PrpC, in the MCC. This inhibition results in attenuated survival in Mtb infected macrophages. Inhibitors are shown in red. This model is derived and modified from Capyk et al. [188].