Mycobacterium tuberculosis (Mtb) secretes proteases and peptidases to subjugate its host. Out of its sixty plus proteases, atleast three are reported to reach host macrophages. In this study, we show that Mtb also delivers a lysyl alanine aminopeptidase, PepN (Rv2467) into host macrophage cytosol. Our comparative in silico analysis shows PepN Mtb highly conserved across all pathogenic mycobacteria. Non-pathogenic mycobacteria including M . smegmatis (Msm) also encode pepN . PepN protein levels in both Mtb (pathogenic) and Msm (non-pathogenic) remain uniform across all in vitro growth phases. Despite such tight maintenance of PepNs’ steady state levels, upon supplementation, Mtb alone allows accumulation of any excessive PepN. In contrast, Msm does not. It not only proteolyzes, but also secretes out the excessive PepN, be it native or foreign. Interestingly, while PepN Mtb is required for modulating virulence in vivo , PepN Msm is essential for Msm growth in vitro . Despite such essentiality difference, both PepN Mtb and PepN Msm harbor almost identical N-terminal M1-type peptidase domains that significantly align in their amino acid sequences and overlap in their secondary structures. Their C-terminal ERAP1_C-like domains however align much more moderately. Our in vitro macrophage-based infection experiments with MtbΔ pepN -expressing pepN Msm reveals PepN Msm also retaining the ability to reach host cytosol. Lastly, but notably, we determined the PepN Mtb and PepN Msm interactomes and found them to barely coincide. While PepN Mtb chiefly interacts with Mtb’s secreted proteins, PepN Msm primarily coimmunoprecipitates with Msm’s housekeeping proteins. Thus, despite high sequence homology and several common properties, our comparative analytical study reveals host-centric traits of pathogenic and bacterial-centric traits of non-pathogenic PepNs.

Dual-emissive carbon dots (D-CDs) are in high demand for multipurpose applications. Herein, nitrogen-doped carbon dots possessing inherent dual emissions at green (490 nm) and yellow (570 nm) are synthesized hydrothermally from 3,4-diaminobenzoic acid and hydrazine hydrate. The relative intensity of the two emission bands is dependent markedly on the pH of medium, making the D-CDs a robust pH sensor within the pH range of 2.0−7.6. The ratiometric emission of D-CDs is sensitive to trace amounts of Fe 3+ ; the 490 nm emission intensity diminishes with simultaneous amplification of the 570 nm emission intensity. A discernible color change from light green to bright yellow is evident under an ultraviolet (UV) lamp with increasing Fe 3+ concentrations, and the CIE color coordinate shifts from (0.33, 0.43) to (0.47, 0.50). Primarily, the complexation between D-CDs and Fe 3+ and the accompanying aggregation play a dominant role in the ratiometric change of the emission intensities. For the on-spot detection of Fe 3+ , smartphone-based and solid-phase sensing is accomplished. The D-CDs also display decent anticancer properties against the breast cancer cell line MCF-7 and effectively detect the intracellular pH and exogenous Fe 3+ inside the MCF-7 cells. KEYWORDS: N-doped carbon dots, ratiometric detection of pH and Fe 3+ , pH and Fe 3+ sensing inside cells, solid-phase and smartphone-based Fe 3+ detection

Drug repurposing presents a workable strategy in tackling antibiotic resistance. Many known drugs have been repurposed for their applications against different targets. Antihistamines that are usually used to treat allergy symptoms can be combined with nanoscale materials to enhance their efficiency. Herein, we explored the antimicrobial properties of a common antihistamine drug, promethazine, in Gram-positive and Gram-negative bacteria. Being positively charged, promethazine was easily incorporated into the mannose-conjugated bovine serum albumin-stabilized promethazine hydrochloride gold nanoclusters. Capping with d-mannose helped in targeting the bacteria by inhibiting their adhesive appendage called pili. Following their uptake, drugs released inside the bacteria caused reactive oxygen species production and membrane permeability alteration, ultimately resulting in bacterial inhibition. Additionally, they were also explored for biofilm eradication. As observed through staining assays, the number of dead cells increased with increasing concentration of drug-loaded gold nanoclusters in the biofilm mass. Therefore, the as-synthesized mannosylated gold nanoclusters incorporated with promethazine were analyzed for potential antibacterial and antibiofilm applications.