25 research outputs found
Design and synthesis of multivalent α-1,2-trimannose-linked bioerodible microparticles for applications in immune response studies of Leishmania major infection
Leishmaniasis, a neglected tropical disease, currently infects approximately 12 million people worldwide with 1 to 2 million new cases each year in predominantly underdeveloped countries. The treatment of the disease is severely underdeveloped due to the ability of the Leishmania pathogen to evade and abate immune responses. In an effort to develop anti-leishmaniasis vaccines and adjuvants, novel carbohydrate-based probes were made to study the mechanisms of immune modulation. In this study, a new bioerodible polyanhydride microparticle was designed and conjugated with a glycodendrimer molecular probe. This molecular probe incorporates a pathogen-like multivalent display of α-1,2-trimannose, for which a more efficient synthesis was designed, with a tethered fluorophore. Further attachment of the glycodendrimer to a biocompatible, surface eroding microparticle allows for targeted uptake and internalization of the pathogen-associated oligosaccharide by phagocytic immune cells. The α-1,2-trimannose-linked bioerodible microparticles were found to be safe after administration into the footpad of mice and demonstrated a similar response to α-1,2-trimannose-coated latex beads during L. major footpad infection. Furthermore, the bioerodible microparticles allowed for investigation of the role of pathogen-associated oligosaccharides for recognition by pathogen-recognition receptors during L. major-induced leishmaniasis
Regulatory and mutational mechanisms for differential expression of the CmeABC multidrug efflux pump in Campylobacter jejuni
Campylobacter jejuni is a zoonotic, foodborne pathogen causing gastroenteritis in humans. The multidrug efflux pump CmeABC plays a key role in antimicrobial resistance by extruding structurally diverse compounds and is essential for intestinal colonization by mediating bile resistance. Expression of cmeABC is under the control of CmeR, a TetR family transcriptional regulator, and CosR, an oxidative stress response regulator. However, the molecular basis and functional consequences of differential CmeABC expression as well as the interactive role of CosR and CmeR in modulating cmeABC expression are still unknown. To address these questions, we performed two sets of studies. In the first study, we evaluated differential expression of cmeABC in naturally occurring C. jejuni isolates and identified the mutations associated with overexpression of cmeABC. It was found that 67% of examined C. jejuni isolates exhibited a CmeABC-overexpressed phenotype as determined by immunoblotting and real-time RT-PCR. This phenotype was further linked to mutations in the cmeABC promoter sequence that decreased the binding of CmeR to the promoter DNA or a reduced cmeR expression. Consequently, both types of mutation increased expression of cmeABC. Additionally, the CmeABC-overexpressed phenotype was associated with increased emergence of ciprofloxacin-resistant mutants in cultures treated with a fluoroquinolone antibiotic.
In the second study, we demonstrated that CmeR and CosR simultaneously bound to two separate sites in the cmeABC promoter, providing dual repression of cmeABC expression. The two regulators interact with the cmeABC promoter independently, but maximal repression by CmeR and CosR requires a 17 bp spacer between the binding sites as shortening the spacer interferes with CmeR binding of the promoter in the presence of CosR. Additionally, we demonstrated that CosR utilizes the single cysteine residue (C218) to sense oxidative stress as oxidation of C218 inhibited CosR binding to the promoter, providing a mechanistic explanation for oxidative-stress-induced, CosR-mediated overexpression of cmeABC.
Together, these results reveal sophisticated mechanisms that modulate expression of cmeABC and identify a new signal (oxidative stress) that interacts with this efflux system. Considering the important role of CmeABC in Campylobacter pathobiology, the diverse mechanisms influencing cmeABC expression may facilitate Campylobacter adaptation to diverse environmental conditions.</p
Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of thecmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABCpromoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression ofcmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.This article is from PLoS ONE 10 (2015): e0131534, doi:10.1371/journal.pone.0131534. Posted with permission.</p
Binding of CmeR to variants of the cmeABC promoter in different isolates.
<p>(A) Sequence alignment of the cmeABC promoter region illustrating the 16-base inverted repeat of the CmeR binding site shown in lowercase letters. The strain names are listed on the left of each sequence. All mutations differing from the 11168 promoter are highlighted in bold. (-) indicates a deleted base. (B) EMSA showing the binding of rCmeRSS to different promoter variants. The control probes include the NCTC 11168 probe (lanes 1–4) in panels I-III and the 81–176 probe (lanes 1–4) in panels IV to VI. The variant promoter probes include CT1:1 (panel I, lanes 5–8), CT1:9 (panel II, lanes 5–8), M32506 (panel III, lanes 5–8), X7199 (panel IV, lanes 5–8), CT3:7 (panel V, lanes 5–8), and CT9:20 (panel VI, lanes 5–8). For each probe, the amount of rCmeRSS used for the each reaction was 0 (lanes 1 and 5), 60 (lanes 2 and 6), 120 (lanes 3 and 7), and 180 ng (lanes 4 and 8), respectively. The rCmeRSS-DNA complexes are indicated with a “C” and the unbound promoter probe is indicated with a “P”.</p
Mutation, expression, and phenotypes of CmeR in selected <i>C</i>. <i>jejuni</i> isolates.
<p>*In relative to the expression level in NCTC 11168 as determined by RT-PCR</p><p>**Isolate is phenotypically classified as WEL</p><p>Mutation, expression, and phenotypes of CmeR in selected <i>C</i>. <i>jejuni</i> isolates.</p
Inability of recombinant CmeR from isolate CT2:2 to bind to the promoter DNA of <i>cmeABC</i>.
<p>(A) Immunoblotting of purified rCmeRSS (lane 2; wild-type CmeR with C69 and C166 replaced with serine) and rCmeR-tr (lane 3; truncated CmeR after residue 193 from isolate CT2:2) with the anti-CmeR antibody. Lane 1 is the protein standard ladder. (B) EMSA showing binding of the 11168 cmeABC promoter by rCmeRSS (lanes 1–4) and rCmeR-tr (lanes 5–8). Proteins were added at 0, 60 (lanes 2 and 6), 120 (lanes 3 and 7), 180 ng (lanes 4 and 8). The locations of protein-DNA complexes and the probe are indicated.</p
Expression of CmeR in various isolates and its correlation with CmeABC expression.
<p>(A) Immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–9), and 11168Δ<i>cmeR</i> (lane 10) with the anti-CmeR antibody. The clinical isolates in lanes 2 to 9 are M63885, CT9:7, CB2:6, CB2:8, CB2:11, S13530, T37957A, and X7199, respectively. (B) Immunoblotting of whole cell proteins from 11168ΔcmeR (lane 1), CT2:2 (lane 2), and NCTC 11168 (lanes 3) with anti-CmeR, anti-CmeB, and anti-CmeA antibodies.</p
Bacterial strains or plasmids used in this study.
<p>Bacterial strains or plasmids used in this study.</p
Effect of various mutations in CmeR and the promoter region on transcription of <i>cmeABC</i>.
<p>The names of the promoters used in the transcriptional fusions and β-galactosidase assays are indicated under each panel. Each promoter was assayed in the wild-type 81–176 background (A) and the 81–176ΔcmeR background (B). The data represent means with standard deviation from three independent experiments. The relative difference in transcription (fold change) due to repression by CmeR for each promoter is shown in (C) and was determined by comparison of transcription in the absence of CmeR (B) to the presence of CmeR (A). The unpaired Student’s t-test with Welch’s correction was used for comparison of the means with significance set at 0.05.</p