PmrA Protein Research Articles

Mechanistic and biophysical characterization of polymyxin resistance response regulator PmrA in Acinetobacter baumannii.

Acinetobacter baumannii PmrAB is a crucial two-component regulatory system (TCS) that plays a vital role in conferring resistance to polymyxin. PmrA, a response regulator belonging to the OmpR/PhoB family, is composed of a C-terminal DNA-binding effector domain and an N-terminal receiver domain. The receiver domain can be phosphorylated by PmrB, a transmembrane sensor histidine kinase that interacts with PmrA. Once phosphorylated, PmrA undergoes a conformational change, resulting in the formation of a symmetric dimer in the receiver domain. This conformational change facilitates the recognition of promoter DNA by the DNA-binding domain of PmrA, leading to the activation of adaptive responses. X-ray crystallography was carried out to solve the structure of PmrA receiver domain. Electrophoretic mobility shift assay and Isothermal titration calorimetry were recruited to validate the interaction between the recombinant PmrA protein and target DNA. Field-emission scanning electron microscopy (FE-SEM) was employed to characterize the surface morphology of A. baumannii in both the PmrA knockout and mutation strains. The receiver domain of PmrA follows the canonical α5β5 response regulator assembly, which undergoes dimerization upon phosphorylation and activation. Beryllium trifluoride is utilized as an aspartate phosphorylation mimic in this process. Mutations involved in phosphorylation and dimerization significantly affected the expression of downstream pmrC and naxD genes. This impact resulted in an enhanced cell surface smoothness with fewer modifications, ultimately contributing to a decrease in colistin (polymyxin E) and polymyxin B resistance. Additionally, a conservative direct-repeat DNA PmrA binding sequence TTTAAGNNNNNTTTAAG was identified at the promoter region of the pmrC and naxD gene. These findings provide structural insights into the PmrA receiver domain and reveal the mechanism of polymyxin resistance, suggesting that PmrA could be a potential drug target to reverse polymyxin resistance in Acinetobacter baumannii.

Acinetobacter baumannii under Acidic Conditions Induces Colistin Resistance through PmrAB Activation and Lipid A Modification.

Colistin is a last-resort antimicrobial agent for treating carbapenem-resistant Acinetobacter baumannii infections. The activation of PmrAB by several environmental signals induces colistin resistance in Gram-negative bacteria. This study investigated the molecular mechanisms of colistin resistance in A. baumannii under acidic conditions using wild-type (WT) A. baumannii 17978, ΔpmrA and ΔpmrB mutants, and pmrA-complemented strains. The pmrA or pmrB deletion did not affect the growth of A. baumannii under acidic or aerobic conditions. A. baumannii under acidic (pH 5.5) and high-iron (1 mM) conditions showed 32- and 8-fold increases in the minimum inhibitory concentrations (MICs) of colistin, respectively. The ΔpmrA and ΔpmrB mutants at pH 5.5 showed a significant decrease in colistin MICs compared to the WT strain at pH 5.5. No difference in colistin MICs was observed between WT and mutant strains under high-iron conditions. The pmrCAB expression significantly increased in the WT strain at pH 5.5 compared to the WT strain at pH 7.0. The pmrC expression significantly decreased in two mutant strains at pH 5.5 compared to the WT strain at pH 5.5. The PmrA protein was expressed in the ΔpmrA strain carrying ppmrA_FLAG plasmids at pH 5.5 but not at pH 7.0. Lipid A modification by the addition of phosphoethanolamine was observed in the WT strain at pH 5.5. In conclusion, this study demonstrated that A. baumannii under acidic conditions induces colistin resistance via the activation of pmrCAB operon and subsequent lipid A modification.

Insights from the molecular docking analysis of colistin with the PmrA protein model from Acinetobacter baumannii.

Acinetobacter baumannii (AB) is one of the most common causes of nosocomial infections. Therefore, it is of interest to design and develop drugs against Acinetobacter baumannii. A strain of AB showing MIC 32 µg/ml against colistin was isolated from a hospital environment in Iran. Hence, we document data to glean insights from the molecular docking analysis of colistin with the PmrA protein from this bacterium.

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate

Background:There is limited information on the 3D prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of PmrA/B in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods:The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and MIC assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results:Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 β-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three β-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 β-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: TheDNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii.

Interaction between LINC-ROR and Stemness State in Gastric Cancer Cells with Helicobacter pylori Infection.

Large intergenic non-coding RNA regulator of reprogramming (LINC-ROR), as a cancer-related Long non-coding RNA, has vital roles in stem cell survival, pluripotency, differentiation, and self-renewal in human embryonic stem cell. However, cancer-related molecular mech¬anisms, its functional roles, and clinical value of LINC-ROR in gastric cancer (GC) remain unclear. In this study, we aimed to investigate probable interplay between LINC-ROR with SALL4 stemness regulator and their role with the development of the disease. The mRNA expression profile of LINC-ROR and SALL4 was assessed in tumoral and adjacent non-cancerous tissues of GC patients, using quantitative real-time PCR. Significant LINC-ROR underexpression and SALL4 overexpression were observed in 55.81% and 75.58% (p < 0.0001) of samples, respectively. The expression of LINC-ROR and SALL4 were significantly correlated with each other (p = 0.044). There was an association between the underexpression of LINC-ROR and sex, stage of tumor progression, tumor type, and location of tumor (p < 0.05), and Helicobacter pylori infection with SALL4 expression (p = 0.036). There were also significant correlations between concomitant mRNA expression of SALL4 and LINC-ROR in tumors located at distal noncardiac, positive for H. pylori infection, tumors with invasion into the muscle layer of the stomach, and grade II tumor (p < 0.05). The clinical results of the SALL4-LINC-ROR association propose a probable functional interaction between these markers in tumor maintenance and aggressiveness. Our study can help to understand one of the mechanisms involved in the progression of gastric cancer through the function of these regulators.

The lipopolysaccharide modification regulator PmrA limitsSalmonellavirulence by repressing the type three-secretion system Spi/Ssa

The regulatory protein PmrA controls expression of lipopolysaccharide (LPS) modification genes in Salmonella enterica serovar Typhimurium, the etiologic agent of human gastroenteritis and murine typhoid fever. PmrA-dependent LPS modifications confer resistance to serum, Fe(3+), and several antimicrobial peptides, suggesting that the pmrA gene is required for Salmonella virulence. We now report that, surprisingly, a pmrA null mutant is actually hypervirulent when inoculated i.p. into C3H/HeN mice. We establish that the PmrA protein binds to the promoter and represses transcription of ssrB, a virulence regulatory gene required for expression of the Spi/Ssa type three-secretion system inside macrophages. The pmrA mutant displayed heightened expression of SsrB-dependent genes and faster Spi/Ssa-dependent macrophage killing than wild-type Salmonella. A mutation in the ssrB promoter that abolished repression by the PmrA protein rendered Salmonella as hypervirulent as the pmrA null mutant. The antivirulence function of the PmrA protein may limit the acute phase of Salmonella infection, thereby enhancing pathogen persistence in host tissues.

An Allele of an Ancestral Transcription Factor Dependent on a Horizontally Acquired Gene Product

Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired pmrD gene product to promote gene expression. We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties.

Ancestral Genes Can Control the Ability of Horizontally Acquired Loci to Confer New Traits

Horizontally acquired genes typically function as autonomous units conferring new abilities when introduced into different species. However, we reasoned that proteins preexisting in an organism might constrain the functionality of a horizontally acquired gene product if it operates on an ancestral pathway. Here, we determine how the horizontally acquired pmrD gene product activates the ancestral PmrA/PmrB two-component system in Salmonella enterica but not in the closely related bacterium Escherichia coli. The Salmonella PmrD protein binds to the phosphorylated PmrA protein (PmrA-P), protecting it from dephosphorylation by the PmrB protein. This results in transcription of PmrA-dependent genes, including those conferring polymyxin B resistance. We now report that the E. coli PmrD protein can activate the PmrA/PmrB system in Salmonella even though it cannot do it in E. coli, suggesting that these two species differ in an additional component controlling PmrA-P levels. We establish that the E. coli PmrB displays higher phosphatase activity towards PmrA-P than the Salmonella PmrB, and we identified a PmrB subdomain responsible for this property. Replacement of the E. coli pmrB gene with the Salmonella homolog was sufficient to render E. coli resistant to polymyxin B under PmrD-inducing conditions. Our findings provide a singular example whereby quantitative differences in the biochemical activities of orthologous ancestral proteins dictate the ability of a horizontally acquired gene product to confer species-specific traits. And they suggest that horizontally acquired genes can potentiate selection at ancestral loci.

Acid pH activation of the PmrA/PmrB two‐component regulatory system of Salmonella enterica

Acid pH often triggers changes in gene expression. However, little is known about the identity of the gene products that sense fluctuations in extracytoplasmic pH. The Gram-negative pathogen Salmonella enterica serovar Typhimurium experiences a number of acidic environments both inside and outside animal hosts. Growth in mild acid (pH 5.8) promotes transcription of genes activated by the response regulator PmrA, but the signalling pathway(s) that mediates this response has thus far remained unexplored. Here we report that this activation requires both PmrA's cognate sensor kinase PmrB, which had been previously shown to respond to Fe3+ and Al3+, and PmrA's post-translational activator PmrD. Substitution of a conserved histidine or of either one of four conserved glutamic acid residues in the periplasmic domain of PmrB severely decreased or abolished the mild acid-promoted transcription of PmrA-activated genes. The PmrA/PmrB system controls lipopolysaccharide modifications mediating resistance to the antibiotic polymyxin B. Wild-type Salmonella grown at pH 5.8 were > 100 000-fold more resistant to polymyxin B than organisms grown at pH 7.7. Our results suggest that protonation of the PmrB periplasmic histidine and/or of the glutamic acid residues activate the PmrA protein, and that mild acid promotes cellular changes resulting in polymyxin B resistance.

Transcriptional Regulation of the 4-Amino-4-deoxy-L-arabinose Biosynthetic Genes in Yersinia pestis

Inducible membrane remodeling is an adaptive mechanism that enables Gram-negative bacteria to resist killing by cationic antimicrobial peptides and to avoid eliciting an immune response. Addition of 4-amino-4-deoxy-l -arabinose (4-aminoarabinose) moieties to the phosphate residues of the lipid A portion of the lipopolysaccharide decreases the net negative charge of the bacterial membrane resulting in protection from the cationic antimicrobial peptide polymyxin B. In Salmonella enterica serovar Typhimurium, the PmrA/PmrB two-component regulatory system governs resistance to polymyxin B by controlling transcription of the 4-aminoarabinose biosynthetic genes. Transcription of PmrA-activated genes is induced by Fe(3+), which is sensed by PmrA cognate sensor PmrB, and by low Mg(2+), in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg(2+)-responding PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein. Surprisingly, Yersinia pestis can promote PhoP-dependent modification of its lipid A with 4-aminoarabinose despite lacking a PmrD protein. Here we report that Yersinia uses different promoters to transcribe the 4-aminoarabinose biosynthetic genes pbgP and ugd depending on the inducing signal. This is accomplished by the presence of distinct binding sites for the PmrA and PhoP proteins in the promoters of the pbgP and ugd genes. Our results demonstrate that closely related bacterial species may use disparate regulatory pathways to control genes encoding conserved proteins.

Signal-dependent Binding of the Response Regulators PhoP and PmrA to Their Target Promoters in Vivo

Low Mg2+ promotes phosphorylation of the response regulators PhoP and PmrA and transcription of their activated genes in Salmonella enterica. Using chromatin immunoprecipitation, we have now determined that the PhoP and PmrA proteins are recruited to the regulatory region of their target genes when bacteria experience low Mg2+ but not when they are grown in high Mg2+. Even when the PhoP protein was artificially produced at 4-fold higher levels than the wild-type strain, promoter occupancy required the low Mg2+ signal. Substitution of the predicted phosphorylation site Asp-52 with a valine residue abolished phosphorylation of the PhoP protein, resulting in loss of PhoP binding to target promoters and transcription of PhoP-activated genes. Our results indicate that the promoter binding ability of the PhoP and PmrA proteins occurring in low Mg2+ is correlated with phosphorylation of these proteins in vivo.

Connecting two-component regulatory systems by a protein that protects a response regulator from dephosphorylation by its cognate sensor.

A fundamental question in signal transduction is how an organism integrates multiple signals into a cellular response. Here we report the mechanism by which the Salmonella PmrA/PmrB two-component system responds to the signal controlling the PhoP/PhoQ two-component system. We establish that the PhoP-activated PmrD protein binds to the phosphorylated form of the response regulator PmrA, preventing both its intrinsic dephosphorylation and that promoted by its cognate sensor kinase PmrB. This results in PmrA-mediated transcription because phosphorylated PmrA exhibits higher affinity for its target promoters than unphosphorylated PmrA. A PmrD-independent form of the PmrA protein was resistant to PmrB-catalyzed dephosphorylation and promoted transcription of PmrA-activated genes in the absence of inducing signals. This is the first example of a protein that enables a two-component system to respond to the signal governing a different two-component system by protecting the phosphorylated form of a response regulator.

Closing the loop: the PmrA/PmrB two-component system negatively controls expression of its posttranscriptional activator PmrD.

A fundamental question in biology is how an organism integrates multiple signals to mediate an appropriate cellular response. The PmrAPmrB two-component system of Salmonella enterica can be activated independently by Fe(3+), which is sensed by the PmrB protein, and in low Mg(2+), which is sensed by the PhoQ protein. The low-Mg(2+) activation requires pmrD, a PhoPPhoQ-activated gene that activates the response regulator PmrA at a posttranscriptional level. We now report that pmrD expression is negatively regulated by the PmrAPmrB system. Conditions that activate the PmrA protein independently of pmrD, such as exposure to Fe(3+), resulted in lower levels of pmrD transcription. The PmrA protein footprinted the pmrD promoter upstream of the PhoP-binding site but did not interfere with binding of the PhoP protein. Mutation of the PmrA-binding site in the pmrD promoter abolished PmrA-mediated repression. Negative regulation of the PhoPPhoQ-activated pmrD gene by the PmrAPmrB system closes a regulatory circuit designed to maintain proper cellular levels of activated PmrA protein and constitutes a singular example of a multicomponent feedback loop.

Cloning of the Aspergillus niger pmrA gene, a homologue of yeast PMR1, and characterization of a pmrA null mutant

The pmrA gene, a yeast PMR1 homologue, was isolated from Aspergillus niger. Sequence analysis of the pmrA cDNA and the genomic DNA revealed that two introns exist in the coding region, and that an open reading frame in the cDNA encodes a polypeptide of 1056 amino acids containing all the conserved regions present in P-type Ca 2+-ATPases. The predicted pmrA protein exhibited a high degree of sequence similarity to the Pmr1 proteins from yeasts and mammalians (50–59% identity). The expression of the pmrA cDNA partially restored the growth defect of Yarrowia lipolytica pmr1 null mutant on EGTA-containing medium. This indicates that the A. niger pmrA gene encodes a functional homologue of the yeast P-type Ca 2+-ATPase involved in the secretory pathway. An A. niger pmrA null mutant exhibited growth retardation on EGTA-containing medium and the growth defect was overcome by adding Ca 2+ or Mn 2+ into the medium. This suggests an involvement of the pmrA protein in Ca 2+ and Mn 2+ homeostasis in A. niger.

Cloning of the Aspergillus niger pmrA gene, a homologue of yeast PMR1, and characterization of a pmrA null mutant.

The pmrA gene, a yeast PMR1 homologue, was isolated from Aspergillus niger. Sequence analysis of the pmrA cDNA and the genomic DNA revealed that two introns exist in the coding region, and that an open reading frame in the cDNA encodes a polypeptide of 1056 amino acids containing all the conserved regions present in P-type Ca2+-ATPases. The predicted pmrA protein exhibited a high degree of sequence similarity to the Pmr1 proteins from yeasts and mammalians (50-59% identity). The expression of the pmrA cDNA partially restored the growth defect of Yarrowia lipolytica pmr1 null mutant on EGTA-containing medium. This indicates that the A. niger pmrA gene encodes a functional homologue of the yeast P-type Ca2+-ATPase involved in the secretory pathway. An A. niger pmrA null mutant exhibited growth retardation on EGTA-containing medium and the growth defect was overcome by adding Ca2+ or Mn2+ into the medium. This suggests an involvement of the pmrA protein in Ca2+ and Mn2+ homeostasis in A. niger.

Molecular Characterization of the PmrA Regulon

The two-component system PmrA/PmrB of Salmonella enterica controls expression of several loci including those mediating modifications in the lipopolysaccharide that result in polymyxin resistance. To gain insight in the regulation of polymyxin resistance, we mapped the transcription start sites of the PmrA-regulated genes pmrC, pmrG, pbgPE, and ugd and identified a conserved sequence in the promoter region of the first three genes. His-tagged PmrA protein could gel shift DNA fragments containing the promoters of the pmrC, pmrG, and pbgPE genes but not the udg promoter. DNase I footprinting analysis of the pmrC, pmrG, and pbgPE promoters indicate that phosphorylated as well as unphosphorylated PmrA bind to a 16-base pair imperfect inverted repeat sequence (5'-TTAAKTTCTTAAKGTT-3'), which is found 40, 80, and 38 nucleotides upstream from the transcription start sites of the pmrC, pmrG, and pbgPE genes, respectively. Our data suggest that a PmrA dimer activates transcription of the divergent pmrG and pbgPE promoters by binding to a single site in the pmrG-pbgPE intergenic region and that the ugd gene is regulated by the PmrA/PmrB system only indirectly.

Two-component regulatory systems can interact to process multiple environmental signals.

The PhoP/PhoQ two-component system of Salmonella typhimurium governs transcription of some 25 loci in response to the extracellular concentration of Mg2+. We have now identified one of these loci as pmrCAB, which codes for a two-component system that mediates resistance to the antibiotic polymyxin B. Transcription of seven of 25 PhoP-activated loci was dependent on a functional PmrA protein, the response regulator of the PmrA/PmrB system. Expression of the PmrA-dependent loci was induced by either Mg2+ limitation or mild acidification, whereas transcription of a PmrA-independent gene was activated by Mg2+ limitation but not acid pH. Induction of PmrA-activated genes by Mg2 limitation required the PhoP and PhoQ proteins. In contrast, the acid-mediated activation of PmrA-regulated genes occurred in strains that were missing either one of these proteins. Transcriptional regulation by a cascade of two-component systems allows pathogenic bacteria to express their virulence determinants in response to a broader spectrum of environmental cues.