ObjectivesTo investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii.MethodsChlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86–299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays.ResultsMIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity.ConclusionsThis study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.
Multidrug efflux pumps are important drivers of antibiotic resistance in Acinetobacter baumannii and other pathogens, however their ‘natural’ roles beyond transport of clinical antimicrobials are poorly described. Polyamines are an ancient class of molecules with broad roles in all three kingdoms of life, and are the likely natural substrate of at least one efflux pump family. We have defined the transcriptome of A. baumannii following treatment with high levels of the polyamines putrescine, cadaverine, spermidine and spermine. These molecules influenced expression of multiple gene classes in A. baumannii including those associated with virulence, and the four polyamines induced distinct but overlapping transcriptional responses. Polyamine shock also induced expression of the MFS-family efflux pump gene amvA and its repressor gene amvR. Loss of amvA dramatically reduced tolerance to the long-chain triaamine spermidine, but caused only modest changes in resistance to known AmvA substrates such as acriflavine. We confirmed reduced accumulation of spermidine in amvA-deficient A. baumannii, and showed that its expression is induced by long-chain polyamines through its cognate regulator AmvR. Our findings suggest that the conserved A. baumannii efflux pump AmvA has evolved to export spermidine from the cell, but that its substrate recognition promiscuity also allows activity against clinically-important biocides and antibiotics.ImportanceAMR genes, including multidrug efflux pumps, evolved long before the ubiquitous use of antimicrobials in medicine and infection control. Multidrug efflux pumps often transport metabolites, signals and host-derived molecules in addition to antibiotics or biocides. Understanding the ancestral physiological roles of multidrug efflux pumps could help to inform the development of strategies to subvert their activity. In this study, we investigated the response of Acinetobacter baumannii to polyamines, a widespread, abundant class of amino acid-derived metabolites, which led us to identify long-chain polyamines as natural substrates of the disinfectant efflux pump AmvA. A second clinically-important efflux pump, AdeABC, also contributed to polyamine tolerance. Our results suggest that the disinfectant resistance capability that allows A. baumannii to survive in hospitals may have evolutionary origins in the transport of polyamine metabolites.
Sickle cell disease is an inherited genetic disorder that causes anemia, pain crises, organ infarction, and infections in 13 million people worldwide. Previous studies have revealed changes in sialic acid levels associated with red blood cell sickling and showed that stressed red blood cells bare surface-exposed clustered terminal mannose structures mediating hemolysis, but detailed glycan structures and anti-glycan antibodies in sickle cell disease remain understudied. Here, we compiled results obtained through lectin arrays, glycan arrays, and mass spectrometry to interrogate red blood cell glycoproteins and glycan-binding proteins found in the plasma of healthy individuals and patients with sickle cell disease and sickle cell trait. Lectin arrays and mass spectrometry revealed an increase in α2,6 sialylation and a decrease in α2,3 sialylation and blood group antigens displayed on red blood cells. Increased binding of proteins to immunogenic asialo and sialyl core 1, Lewis A, and Lewis Y structures was observed in plasma from patients with sickle cell disease, suggesting a heightened anti-glycan immune response. Data modeling affirmed glycan expression and plasma protein binding changes in sickle cell disease but additionally revealed further changes in ABO blood group expression. Our data provide detailed insights into glycan changes associated with sickle cell disease and refer glycans as potential therapeutic targets.
With new strains of Acinetobacter baumannii undergoing genomic analysis, it has been possible to define regions of genomic plasticity (RGPs), encoding specific adaptive elements. For a selected RGP from a community-derived isolate of A. baumannii, we outline sequences compatible with biosynthetic machinery of surface polysaccharides, specifically enzymes utilized in the dehydration and conversion of UDP-N-acetyl-D-glucosamine (UDP-D-GlcNAc). We have determined the crystal structure of one of these, the epimerase Ab-WbjB. This dehydratase belongs to the ‘extended’ short-chain dehydrogenase/reductase (SDR) family, related in fold to previously characterised enzymes CapE and FlaA1. Our 2.65Å resolution structure of Ab-WbjB shows a hexamer, organised into a trimer of chain pairs, with coenzyme NADP+ occupying each chain. Specific active-site interactions between each coenzyme and a lysine quaternary group of a neighbouring chain interconnect adjacent dimers, so stabilising the hexameric form. We show UDP-GlcNAc to be a specific substrate for Ab-WbjB, with binding evident by ITC (Ka = 0.23 μmol-1). The sequence of Ab-WbjB shows variation from the consensus active-site motifs of many SDR enzymes, demonstrating a likely catalytic role for a specific threonine sidechain (as an alternative to tyrosine) in the canonical active site chemistry of these epimerases.
Serological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.
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