Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.
Methods: The virtual colony count (VCC) ...microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.
Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10
5 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10
7 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA.
Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations, conditions where the antimicrobial agent alone is relatively ineffective.
The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins and LL-37 against biosafety level (BSL)-1 ...and BSL-2 bacteria including
Escherichia coli,
Staphylococcus aureus,
Bacillus cereus, and
Enterobacter aerogenes. In addition, a modified pipetting technique was presented in a 2011 study of defensin activity against the BSL-3 pathogen
Bacillus anthracis. Both studies were published in the journal Antimicrobial Agents and Chemotherapy. Here I report that the method can also detect cross-contamination caused by aerosols utilizing the VCC method of data analysis by quantitative growth kinetics (QGK). The QGK threshold time, or T
t, equivalent to the cycle time C
t reported in 1996 by Heid et al., precisely identifies when wells were inoculated.
Dark blue rings and circles emerged when the non-specific polysaccharide stain lactophenol cotton blue was added to Gram stained slides. The dark blue staining is attributable to the presence of ...capsular polysaccharides and bacterial slime associated with clumps of Gram-negative bacteria. Since all bacterial cells are glycosylated and concentrate polysaccharides from the media, the majority of cells stain light blue. The contrast between dark and light staining is sufficient to enable a digital image processing thresholding technique to be quantitative with little background noise. Prior to the addition of lactophenol cotton blue, the Gram-stained slides appeared unremarkable, lacking ubiquitous clumps or stained polysaccharides. Adding lactophenol cotton blue to Gram stained slides is a quick and inexpensive way to screen cell cultures for bacterial slime, clumps and biofilms that are invisible using the Gram stain alone.
Dengue virus (DENV) infection is a serious public health threat worldwide that demands effective treatment. In the search for potent virus protease inhibitors, several cone snail venoms were screened ...against serotype 2 DENV NS2B-NS3 protease, and one conotoxin, MrIA, was identified to have inhibitory activity. The inhibitory activity was attributed to a disulfide bond-mediated loop, from which rational optimization was made to improve the potency and stability. An eight-residue cyclic peptide inhibitor was finally obtained with high potency (inhibitory constant 2.2 μM), stability, and cell permeability. This inhibitor can thus serve as a good lead for DENV drug development. In addition, this work highlights the critical effect of peptide cyclization on the potency of oligopeptide inhibitors against DENV protease, which may advance the design of peptide inhibitors for homologous virus proteases.
Despite the small size and conserved tertiary structure of defensins, little is known at a molecular level about the basis of their functional versatility. For insight into the mechanism(s) of ...defensin function, we prepared enantiomeric pairs of four human defensins, HNP1, HNP4, HD5, and HBD2, and studied their killing of bacteria, inhibition of anthrax lethal factor, and binding to HIV-1 gp120. Unstructured HNP1, HD5, and HBD3 and several other human α- and β-defensins were also examined. Crystallographic analysis showed a plane of symmetry that related LHNP1 and DHNP1 to each other. Either d-enantiomerization or linearization significantly impaired the ability of HNP1 and HD5 to kill Staphylococcus aureus but not Escherichia coli. In contrast, LHNP4 and DHNP4 were equally bactericidal against both bacteria. d-Enantiomers were generally weaker inhibitors or binders of lethal factor and gp120 than their respective native, all-l forms, although activity differences were modest, particularly for HNP4. A strong correlation existed among these different functions. Our data indicate: (a) that HNP1 and HD5 kill E. coli by a process that is mechanistically distinct from their actions that kill S. aureus and (b) that chiral molecular recognition is not a stringent prerequisite for other functions of these defensins, including their ability to inhibit lethal factor and bind gp120 of HIV-1.
Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.
Methods: The virtual colony count (VCC) ...microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.
Results: Adding tRNA 1:1 to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10
5 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10
7 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA.
Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations and biofilms, conditions where the antimicrobial agent alone is relatively ineffective.
Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.
Methods: The virtual colony count (VCC) ...microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.
Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10
5 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10
7 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA.
Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations and biofilms, conditions where the antimicrobial agent alone is relatively ineffective.
Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly ...conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.
HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr¹⁶, Ile²⁰, Leu²⁵, and Phe²⁸. Previously, alanine scanning mutagenesis identified each of ...these residues and other hydrophobic residues as important for function. Here we report further structural and functional studies of residues shown to interact with one another across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested binding to HIV-1 gp120 and HNP1 by surface plasmon resonance, binding to HIV-1 gp41 and HNP1 by fluorescence polarization, inhibition of anthrax lethal factor, and antibacterial activity using the virtual colony count assay. Similar to the previously described single mutant W26A-HNP1, the quadruple mutant displayed the least activity in all functional assays, followed by the double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A single mutations were milder than the double mutant I20A/L25A-HNP1. Crystallographic studies confirmed the correct folding and disulfide pairing, and depicted an array of dimeric and tetrameric structures. These results indicate that side chain hydrophobicity is the critical factor that determines activity at these positions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK