<em>Bacillus subtilis</em> as a Platform for Molecular Characterisation of Regulatory Mechanisms of <em>Enterococcus faecalis</em> Resistance against Cell Wall Antibiotics

To combat antibiotic resistance of Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanSB-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators

Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches

Distinct, IgG1-driven antibody response landscapes demarcate individuals with broadly HIV-1 neutralizing activity

Understanding pathways that promote HIV-1 broadly neutralizing antibody (bnAb) induction is crucial to advance bnAb-based vaccines. We recently demarcated host, viral, and disease parameters associated with bnAb development in a large HIV-1 cohort screen. By establishing comprehensive antibody signatures based on IgG1, IgG2, and IgG3 activity to 13 HIV-1 antigens in 4,281 individuals in the same cohort, we now show that the same four parameters that are significantly linked with neutralization breadth, namely viral load, infection length, viral diversity, and ethnicity, also strongly influence HIV-1-binding antibody responses. However, the effects proved selective, shaping binding antibody responses in an antigen and IgG subclass-dependent manner. IgG response landscapes in bnAb inducers indicated a differentially regulated, IgG1-driven HIV-1 antigen response, and IgG1 binding of the BG505 SOSIP trimer proved the best predictor of HIV-1 neutralization breadth in plasma. Our findings emphasize the need to unravel immune modulators that underlie the differentially regulated IgG response in bnAb inducers to guide vaccine development

Bacillus subtilis as a platform for molecular characterisation of regulatory mechanisms of Enterococcus faecalis resistance against cell wall antibiotics

To combat antibiotic resistance of Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanS B-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators.</p

Time-resolved induction of P<i>_bcrA</i> by bacitracin in an unmarked, sensitive <i>B. subtilis</i> recipient strain.

<p>SGB274, carrying unmarked deletion of <i>bceRS-bceAB</i>, <i>psdRS-psdAB</i>, <i>yxdJK-yxdLM-yxeA</i> and harbouring the P<i>_bcrA</i>-<i>lux</i> reporter construct pNTlux101 and <i>bcrR</i> expression construct was grown in the presence of 0.2% (w/v) xylose (panels A and B), or in the absence of xylose (panels C and D). In early exponential phase (t = 0 min), bacitracin was added to final concentrations of 0 (open circles) 0.03 μg ml⁻¹ (open squares), 0.1 μg ml⁻¹ (grey circles), 0.3 μg ml⁻¹ (solid circles) or 1 μg ml⁻¹ (solid squares), and luminescence normalized to optical density (RLU/OD) was monitored. (A, C) Time-course of promoter induction over 60 min after bacitracin-challenge. (B, D) Dose-response at 30 min post-induction; the time point is labelled with the arrow in the panels above. Results are shown as the mean and standard deviation of three biological replicates.</p

Plasmids and strains used in this study.

a<p>Bac, bacitracin; cm, chloramphenicol; fs, fusidic acid; kan, kanamycin; mls, macrolide-lincosamide-streptogramin B group antibiotics; rif, rifampin; spc, spectinomycin; van, vancomycin; r, resistant.</p

Antibiotic susceptibility of <i>B. subtilis</i> strains.

a<p>Minimal inhibitory concentrations (MIC) determined from three biological replicates; where a range of concentrations is given, results varied between replicates.</p

Primers used in this study.

a<p>Restriction sites are underlined; overlaps to other primers for PCR fusions are shown by lower case letters.</p

VanS_B-VanR_B-dependent induction of P<i>_vanYB</i> by vancomycin in <i>B. subtilis</i>.

<p>Both the P<i>_vanYB</i>-<i>lux</i> reporter construct pCF133 and the P<i>_xylA</i>-<i>vanR_BS_B</i> expression construct pCF132 were introduced into <i>B. subtilis</i> strain TMB1518 (unmarked deletion of <i>bceRS-bceAB</i>, <i>psdRS-psdAB</i>, <i>yxdJK-yxdLM-yxeA</i>). Cultures growing exponentially either (A) in the absence of xylose or (B) in the presence of 0.2% (w/v) xylose were challenged at t = 0 min with 0.01 μg ml⁻¹ (open squares), 0.025 μg ml⁻¹ (grey circles), 0.05 μg ml⁻¹ (solid circles), 0.25 μg ml⁻¹ (solid squares) vancomycin, or left untreated (open circles). Luminescence normalized to optical density (RLU/OD) was monitored over 60 min. Results are shown as the mean and standard deviation of three biological replicates.</p