EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde.

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host-pathogen interactions.

Two Mutations Commonly Associated with Daptomycin Resistance in Enterococcus faecium LiaST120A and LiaRW73C Appear To Function Epistatically in LiaFSR Signaling.

The cyclic antimicrobial lipopeptide daptomycin is now frequently used as a first-line therapy in serious infections caused by multidrug-resistant Enterococcus faecium. Resistance to daptomycin in E. faecium is mediated by activation of the LiaFSR membrane stress response pathway. Deletion of liaR, encoding the response regulator of the system, restores susceptibility to daptomycin, suggesting that the LiaFSR pathway is a potential target for the development of drugs that would induce hypersusceptibility to daptomycin and make it more difficult for enterococci to become daptomycin-resistant. In clinical isolates of E. faecium, substitutions in the membrane-bound histidine kinase LiaS (T120A) and its response regulator LiaR (W73C) are found together, suggesting a potential epistatic relationship in daptomycin resistance. Using in vitro phosphorylation studies, we show that while the phosphotransfer rate of wild-type LiaS and LiaST120A to either wild-type LiaR or LiaRW73C remains rapid and comparable, the LiaS-dependent dephosphorylation rate of phosphorylated LiaRW73C is markedly higher. When the two adaptive mutants LiaRW73C and LiaST210A are paired, however, LiaS-mediated LiaR dephosphorylation is restored back to wild-type levels. Taken together with earlier work showing that LiaRW73C leads to an increased level of oligomerization and subsequently favors an increased level of transcription of the LiaFSR regulon, the net effect of the two commonly found LiaST120A and LiaRW73C alleles would be to coordinately increase the strength and persistence of LiaFSR signaling and decrease daptomycin susceptibility. The in vitro approaches developed in this work also provide the basis for screens for identifying drug candidates that inhibit the LiaFSR pathway.

A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci.

Substitutions in the LiaFSR membrane stress pathway are frequently associated with the emergence of antimicrobial peptide resistance in both Enterococcus faecalis and Enterococcus faecium Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology, including stress responses. We have previously identified a mutation in a gene (designated yybT) in E. faecalis that was associated with the development of daptomycin resistance, resulting in a change at position 440 (yybTI440S) in the predicted protein. Here, we show that intracellular c-di-AMP signaling is present in enterococci, and on the basis of in vitro physicochemical characterization, we show that E. faecalisyybT encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5'pApA. The E. faecalis GdpPI440S substitution reduces c-di-AMP phosphodiesterase activity more than 11-fold, leading to further increases in c-di-AMP levels. Additionally, deletions of liaR (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both E. faecalis and E. faecium also resulted in increased c-di-AMP levels, suggesting that changes in the LiaFSR stress response pathway are linked to broader physiological changes. Taken together, our data show that modulation of c-di-AMP pools is strongly associated with antibiotic-induced cell membrane stress responses via changes in GdpP activity or signaling through the LiaFSR system.

Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance.

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.

The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated E. faecium LiaR protein at 3.2Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.

A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin.

LiaR is a 'master regulator' of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR(D191N) increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.

Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis.

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

Biochemical characterization of cardiolipin synthase mutations associated with daptomycin resistance in enterococci.

Daptomycin (DAP) resistance in enterococci has been linked to mutations in genes that alter the cell envelope stress response (CESR) (liaFSR) and changes in enzymes that directly affect phospholipid homeostasis, and these changes may alter membrane composition, such as that of cardiolipin synthase (Cls). While Cls substitutions are observed in response to DAP therapy, the effect of these mutations on Cls activity remains obscure. We have expressed, purified, and characterized Cls enzymes from both Enterococcus faecium S447 (residues 52 to 482; Cls447a) and Enterococcus faecalis S613 (residues 53 to 483; Cls613a) as well as Cls variants harboring a single-amino-acid change derived from DAP-resistant isolates of E. faecium. E. faecium Cls447a and E. faecalis Cls613a are tightly associated with the membrane and copurify with their substrate, phosphatidylglycerol (PG), and product, cardiolipin (CL). The amount of PG that copurifies with Cls is in molar excess to protein, suggesting that the enzyme localizes to PG-rich membrane regions. Both Cls447a(H215R) and Cls447a(R218Q) showed an increase in V(max) (µM CL/min/µM protein) from 0.16 ± 0.01 to 0.26 ± 0.02 and 0.26 ± 0.04, respectively, indicating that mutations associated with adaptation to DAP increase Cls activity. Modeling of Cls447a to Streptomyces sp. phospholipase D indicates that the adaptive mutations Cls447a(H215R) and Cls447a(R218Q) are proximal to the phospholipase domain 1 (PLD1) active site and near the putative nucleophile H217. As mutations to Cls are part of a larger genomic adaptation process, increased Cls activity is likely to be highly epistatic with other changes to facilitate DAP resistance.

The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design.

BACKGROUND: Streptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens. RESULTS: Here we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3121 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = ß = 90° and γ = 120°. Structural comparisons show that AlrSP shares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway. CONCLUSIONS: We have solved the structure of AlrSP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.