Bacteria possess numerous peptide transporters for importing peptides as nutrients. However, these peptide transporters are now consistently reported to play a role in the virulence of various bacterial pathogens. Their ability to transport peptides has implications in antibacterial therapy as well. Therefore, it would be instrumental to have complete knowledge about the role of peptide transporters in mediating this cross connection between metabolism and pathogenesis. Studies on various peptide transporters in bacterial pathogens have improved our understanding of this field. In this review, we have given an overview of the functioning of bacterial peptide transporters and their contribution in virulence of major bacterial pathogens.
Table of Contents Chemicals and Methods S3 Characterisation of nanoparticles S4 POPG hydrolysis by MALDI-Mass Analysis S7 Phosphatidylcholine hydrolysis UV-Vis assay scheme S8 UV-Visible analysis of p-Nitrophenylphosphatidyl choline (NPPC) hydrolysis S9 Kinetic parameters of NPPC hydrolysis S10 31 P NMR analysis of NPPC hydrolysis S11 XPS and HRTEM analysis of nanoparticles S12 Phosphatidylcholine hydrolysis by nanoceria S13 Confocal study of bacterial cell membrane disruption S13 CFU analysis of Salmonella growth and death curve S15 Confocal microscopy analysis of antibacterial activity S17 Study of pre-attachment phase of biofilm formation S18 Synthesis and characterization of FITC-tagged PAA-Cnp S18 Localization of PAA-Cnp inside biofilm S21 Dispersibility of nanozymes in solution S21 SEM images of biofilm formation/disruption S22 Comparison of biofilm inhibition using oxidase-mimetic S22 Antibacterial activity on various pathogenic bacteria S23 Antibiofouling studies on urinary catheters S28 MIC quantification and comparison S29 Cytotoxicity study against HeLa cells S30 S3 1. Chemicals Cerium chloride heptahydrate (CeCl3.7H2O) was purchased from Avra synthesis Pvt. Ltd. Cerric ammonium nitrate ((NH4)2Ce(NO3)6), ethylenediamine, hydrazine hydrate and ammonium hydroxide, 3-aminopropyltriethoxysilane used for various synthesis were purchased from Sisco Research Laboratories. Sodium polyacrylate and flouresceine isothiocynate used for preparing PAA-Cnp and FITC-tagged PAA-Cnp nanoparticles respectively were purchased from Sigma Aldrich. Trizma base and phosphotidyl choline (lecithin) used for hydrolysis assays were also purchased from Sigma Aldrich. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol and pnitrophenyl phosphorylcholine used for other hydrolysis assays were purchased from TCI Co. Ltd. and Alfa Aesar Fischer Scientific respectively. 2. Characterization Methods Scanning electron microscopy (SEM) and EDX spectrawere performed on a Carl-Zeiss Ultra 55, FEI Sirion UHR SEM and ESEM-Quanta instruments,respectively. Transmission electron microscopy (TEM), high resolution TEM (HRTEM), electron diffraction X-Ray (EDX) analysis and selected area electron diffraction (SAED) were recorded on JEOL transmission electron microscope operating at 200 kV after casting a drop of nanoparticle dispersion in acetone over Cu grid. Powder XRDwas recordedon Philips PANalytical X-ray diffractometer by using a Cu-Kα (1.5406 Å)radiation. X-ray photoelectron spectroscopy (XPS) was acquired on AXIS ULTRA, KRATOS ANALYTICAL, SHIMADAZU. Raman spectroscopy was performed on HORIBA JOBIN YVON LabRAM HR Raman spectrometer (532 nm laser. FT-IR spectra were recorded on aPerkinElmer FT-IR spectrometer. Zeta potential was measured usingMalvern Zetasizer Nano UK.UV-Vis absorption spectra were acquired onSHIMADAZU UV-2600 spectrophotometer. Fluorescence spectroscopy was measured by using HORIBA JOBIN YVON (Fluoromax-4 Spectrofluorometer) instrument. All the 31 P NMR spectra were recorded using AV400 and AV500 MHz Avance Bruker High Resolution...
Salmonella being a successful pathogen, employs a plethora of immune evasion mechanisms. This contributes to pathogenesis, persistence and also limits the efficacy of available treatment. All these contributing factors call upon for new drug targets against Salmonella. For the first time, we have demonstrated that Salmonella upregulates sirtuin 2 (SIRT2), an NAD+ dependent deacetylase in dendritic cells (DC). SIRT2 upregulation results in translocation of NFκB p65 to the nucleus. This further upregulates NOS2 transcription and nitric oxide (NO) production. NO subsequently shows antibacterial activity and suppresses T cell proliferation. NOS2 mediated effect of SIRT2 is further validated by the absence of effect of SIRT2 inhibition in NOS2-/- mice. Inhibition of SIRT2 increases intracellular survival of the pathogen and enhances antigen presentation in vitro. However, in vivo SIRT2 inhibition shows lower bacterial organ burden and reduced tissue damage. SIRT2 knockout mice also demonstrate reduced bacterial organ burden compared to wild-type mice. Collectively, our results prove the role of SIRT2 in Salmonella pathogenesis and the mechanism of action. This can aid in designing of host-targeted therapeutics directed towards inhibition of SIRT2.
Salmonella is a facultative intracellular pathogen that has co-evolved with its host and has also developed various strategies to evade the host immune responses. Salmonella recruits an array of virulence factors to escape from host defense mechanisms. Previously chitinase A (chiA) was found to be upregulated in intracellular Salmonella. Although studies show that several structurally similar chitinases and chitin-binding proteins (CBP) of many human pathogens have a profound role in various aspects of pathogenesis, like adhesion, virulence, and immune evasion, the role of chitinase in the intravacuolar pathogen Salmonella has not yet been elucidated. Therefore, we made chromosomal deletions of the chitinase encoding gene (chiA) to study the role of chitinase of Salmonella enterica in the pathogenesis of the serovars, Typhimurium, and Typhi using in vitro cell culture model and two different in vivo hosts. Our data indicate that ChiA removes the terminal sialic acid moiety from the host cell surface, and facilitates the invasion of the pathogen into the epithelial cells. Interestingly we found that the mutant bacteria also quit the Salmonella-containing vacuole and hyper-proliferate in the cytoplasm of the epithelial cells. Further, we found that ChiA aids in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production in the phagocytes, leading to MHCII downregulation followed by suppression of antigen presentation and antibacterial responses. Notably, in the murine host, the mutant shows compromised virulence, leading to immune activation and pathogen clearance. In continuation of the study in C. elegans, Salmonella Typhi ChiA was found to facilitate bacterial attachment to the intestinal epithelium, intestinal colonization, and persistence by downregulating antimicrobial peptides. This study provides new insights on chitinase as an important and novel virulence determinant that helps in immune evasion and increased pathogenesis of Salmonella.
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