AMPing Up the Pressure: Cell Envelope Signaling Protects Enterococcus faecalis From Antimicrobial Peptides

For many bacteria, the gastrointestinal (GI) tract is a difficult environment to survive in. Even though nutrients are abundant, competition for them is fierce. Bacteria produce a variety of antimicrobial peptides (AMPs), called bacteriocins, that help them establish and protect their niche within the GI microbiome. At the same time, the mammalian immune system also produces AMPs that have a wide range of inhibitory activities against bacteria, fungi, parasites, and viruses. One type of bacteria that can take up residence in the GI tract is enterococci, which are gram-positive cocci that are intrinsically hardy, meaning that they can survive in harsh environments [1]. Enterococci, despite naturally residing in the GI tract, are also opportunistic pathogens capable of leveraging their intrinsic antibiotic resistance and acquiring new resistance genes to cause a range of extraintestinal infections in immunocompromised and critically ill patients [2, 3]. Among the species in the Enterococcus genus, Enterococcus faecalis causes a large number of these infections and is a frequent cause of biofilm-associated infections such as urinary tract infections and endocarditis [4, 5].

Daptomycin is a cyclic lipopeptide antibiotic that is often prescribed to treat antibiotic-resistant enterococcal infections. Daptomycin requires and complexes with calcium ions to fully exhibit its antimicrobial activity. The resulting positively charged daptomycin:Ca²⁺ complex then oligomerizes and inserts itself into bacterial cell membranes, leading to inhibition of cell wall biosynthesis, membrane depolarization, and cell death [6, 7]. Daptomycin resembles AMPs in both its structure and mechanism of action. Because of this, understanding mechanisms of intrinsic and acquired daptomycin resistance can also shed light on enterococcal resistance to other cationic AMPs, which enterococci are often exposed to in the GI tract [8]. Cationic AMPs, such as cathelicidin LL-37, human β-defensin 3 (HBD3), and α-defensin human neutrophil peptide-1 (HNP1), are part of the mammalian innate immune system (Figure 1). Elucidating intrinsic mechanisms of E faecalis resistance against AMPs can help us better understand why this organism is such a successful opportunistic pathogen and may also reveal new evolutionary routes for resistance to daptomycin, an important antibiotic for the treatment of enterococcal infections.

Figure 1.

Cationic antimicrobial peptide structures and characteristics. A, Monomer of human cathelicidin LL-37, from Protein Data Bank (PDB) (ID: 2K6O) [9]. B, Monomer of human β-defensin 3, from PDB (ID: 1KJ6) [10]. C, Monomer of α-defensin human neutrophil peptide-1, from PDB (ID: 2PM5) [11]. Structures were created with Mol* Viewer [12].

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Unlike vancomycin resistance, which depends on the presence of a multigene operon, daptomycin resistance in enterococci can occur through mutations in multiple genes and pathways [13–15]. Previous studies have shown that daptomycin resistance in enterococci occurs primarily through mutations to the LiaFSR system, a 3-component system that regulates the cell-envelope stress response [13, 16]. Secondary mutations in genes such as the dlt operon (which performs D-alanylation of lipoteichoic acid), mprF (which synthesizes lysyl-phosphatidylglycerol), and genes involved in cell membrane phospholipid synthesis also contribute to daptomycin resistance [13, 17–19]. Ultimately, these mutations decrease daptomycin binding to the cell membrane by causing alterations in membrane lipid composition, structure, and arrangement, as well as increasing the cell surface charge. Blocking the LiaFSR system has been proposed as a potential therapeutic strategy because of the critical role that this system plays in modulating daptomycin resistance in enterococci.

The study by Miller at al in this issue of The Journal of Infectious Diseases investigates the regulatory network and role of a LiaFSR-independent daptomycin resistance pathway [20]. Previous work by the same researchers identified a new evolutionary route to daptomycin resistance in E faecalis through mutations in the MadRS (membrane antimicrobial peptide defense) 2-component system [21]. In this prior study, the researchers found that activation of the MadRS system through a mutation in MadS (MadS_A202E), combined with truncation of a putative fatty acid kinase called dak, conferred daptomycin resistance in the absence of LiaFSR signaling. Following up on their initial discovery of the MadRS system's role in daptomycin resistance, in their current study, Miller et al explore the broader regulatory network of MadRS and its role in the cell envelope stress response [20, 21]. Following a series of comprehensive experiments, they propose a model in which the presence of cationic AMPs or antibiotics that resemble AMPs, like daptomycin and bacitracin, triggers the activation of the MadS sensor histidine kinase, which then phosphorylates the response regulator MadR. In addition to cationic AMPs, the researchers also suggest that the MadRS regulon can be primed by dak deletion [20]. Combining RNA-seq analyses and reverse-transcription polymerase chain reaction, they find that activation of MadR leads to upregulation of many genes, including known effectors like the Dlt operon and MprF2, as well as new effectors such as MadEFG, MadLM, and SalA, a putative peptidoglycan hydrolase [20].

The MadRS 2-component system (locus tags EF0926 and EF0927 in the E faecalis V583 strain genome) regulates 2 ATP-binding cassette transporters, EF2752–EF2751 (renamed MadAB) and EF2050–EF2049 (renamed MadLM). These transporters have been shown previously to sense and mediate resistance to bacitracin, a cyclic polypeptide antibiotic [22]. In addition, transposon inactivation of MadRS and MadAB in a prior transposon-insertion sequencing (Tn-seq) study was associated with increased susceptibility to polymyxin, a cyclic cationic AMP [23]. These prior studies implicate the MadRS regulatory network in E faecalis defense against cationic AMP-like antibiotics. Interestingly, Miller et al found that protection against different AMPs and AMP-like antibiotics is mediated by different targets within the MadRS regulon. For example, they show that MadLM provides protection against bacitracin, which is consistent with prior literature [20, 22]. In contrast, protection against daptomycin is not provided by MadEFG nor by MadLM. Instead, MprF2 and the Dlt operon appear to play a crucial role in protection against daptomycin, which is also consistent with prior literature [13, 20, 24]. In the same vein, protection against the host-derived cathelicidin LL-37 depends solely on the MadEFG operon. In addition to LL-37, MadEFG also affects E faecalis survival against HBD3, a phenotype that is seen in a dak deletion mutant isolate [20]. However, the mechanism by which changes in membrane lipid profiles caused by dak deletion interfere with HBD3 activity remains to be elucidated. Interestingly, the MadRS regulon does not appear to affect susceptibility to the cationic AMP HNP1, further highlighting the substrate-specific nature of MadRS activity. Despite being categorized as cationic AMPs, several key structural differences exist between LL-37, HBD3, and HNP1 (Figure 1). Variance in the size, charge, structure, and dimerization of each AMP could influence how the MadRS system recognizes and responds to these peptides, leading to substrate-specific activity.

Miller et al further show that E faecalis defense against AMPs through MadRS signaling extends beyond the in vitro laboratory setting [20]. MadRS is able to protect E faecalis from cationic AMPs produced by Caenorhabditis elegans, and this protection is specific to host-derived cationic AMPs. In a C elegans infection model, Miller et al show that an E faecalis ΔmadR mutant strain is less virulent, leading to higher C elegans survival when compared to an E faecalis strain with intact MadR. Furthermore, this difference in survival is abolished when the same strains are used to infect C elegans that are AMP-deficient. Miller et al also test the protection afforded by MadRS against host-derived cationic AMPs in a mouse peritonitis model, where E faecalis with hyperactivated MadRS is shown to cause more and larger cardiac microlesions compared to E faecalis with baseline MadRS expression [20]. Taken together, these data suggest that the MadRS system protects E faecalis from the host immune response in vivo.

Overall, the investigation performed by Miller et al suggests that the expression of the MadRS regulatory network is crucial for the survival and colonization of E faecalis in the host GI tract, where AMPs are prevalent. Moreover, the MadRS signaling system likely contributes to E faecalis translocation and infection in immunocompromised patients. Further studies should delve into the molecular mechanism by which MadEFG defends E faecalis against the antimicrobial activity of host AMPs like LL-37 and HBD3, and why different effectors in the MadRS regulon respond to different AMPs and antibiotics that resemble them. While Miller et al hypothesize that the MadEFG operon could function similarly to the Streptococcus pneumoniae MacAB-like efflux pump in pumping out LL-37, for the moment this remains a compelling hypothesis in need of testing [25]. Such studies, combined with the insights presented in the current article, will expand our understanding of the intricate pathways, regulators, and effectors that E faecalis employs to withstand the cell membrane–disrupting activity of AMPs and cause disease in susceptible hosts.

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