Reduction of Human Defensin 5 Affords a High-Affinity Zinc-Chelating Peptide

Human defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits three disulfide bonds in the oxidized form (HD5[subscript ox]). It is abundant in small intestinal Paneth cells, which release HD5 into the intestinal lumen and house a labile Zn(II) store of unknown function. Here, we consider the redox properties of HD5 and report that the reduced form, HD5[subscript red], is a metal-ion chelator. HD5 has a midpoint potential of −257 mV at pH 7.0. HD5[subscript red] utilizes its cysteine residues to coordinate one equivalent of Zn(II) with an apparent K[subscript d1] value in the midpicomolar range. Zn(II) or Cd(II) binding perturbs the oxidative folding pathway of HD5[subscript red] to HD5[subscript ox]. Whereas HD5[subscript red] is highly susceptible to proteolytic degradation, the Zn(II)-bound form displays resistance to hydrolytic breakdown by trypsin and other proteases. The ability of a reduced defensin peptide to coordinate Zn(II) provides a putative mechanism for how these peptides persist in vivo.National Institutes of Health (U.S.) (Grant DP2OD007045)Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Dept. of Chemistr

Differences in Fecal Bacterial Community Composition Between Beef Steers which are High-Shedders and Low-Shedders of Shiga Toxin-Producing \u3ci\u3eEscherichia coli\u3c/i\u3e (STEC)

The community composition of the fecal microbiota was compared between beef steers which were high-shedders and low-shedders of Shiga toxin-producing Escherichia coli. Based on Shannon and Chao 1 diversity indices, the high-shedders had a more diverse fecal bacterial community than the low-shedding steers. Members of the genus Prevotella were observed as being more abundant in the low-shedders compared to the high-shedders, while Succinivibrio were more abundant in the high-shedders. Isolation of specific bacteria which are significantly more abundant in low-shedders may pave the way to developing direct-fed microbials which are effective in reducing STEC shedding among high-shedding beef steers

The Effect of Commensal Microbial Communities on the Fecal Shedding of Shiga Toxin-Producing \u3ci\u3eE. coli\u3c/i\u3e (STEC) in Beef Cattle

This ongoing study compares the gut microbial community composition between shedding steers high in shiga toxin-producing E. coli (STEC) counts and low-shedding steers. Shedders were identified among 170 beef animals over three time periods using selective microbiological culture media. The isolated bacterial cultures were confirmed to be STEC using PCR, 16s rRNA sequencing and a shiga toxin immunoassay. The most abundant strains found in the cattle feces were those belonging to the serogroups O111 (40.3%) and O157:H7 (37.3%), with O103 (8.3%), O26 (6.0%), O83 (4.5%), and O55 (3.0%) being detected in much lower numbers . Out of the 52 animals which were identified as super-shedders of STECs which were selected for microbial community analysis, 61.54% shed STEC in at least two of the three sampling time points. Currently, work is being carried out to evaluate the microbial community composition of the identified STEC high-shedding and low-shedding cattle populations using 454-pyrosequencing

Reduction of Human Defensin 5 Affords a High-Affinity Zinc-Chelating Peptide

Human defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits three disulfide bonds in the oxidized form (HD5_ox). It is abundant in small intestinal Paneth cells, which release HD5 into the intestinal lumen and house a labile Zn­(II) store of unknown function. Here, we consider the redox properties of HD5 and report that the reduced form, HD5_red, is a metal-ion chelator. HD5 has a midpoint potential of −257 mV at pH 7.0. HD5_red utilizes its cysteine residues to coordinate one equivalent of Zn­(II) with an apparent <i>K</i>_d1 value in the midpicomolar range. Zn­(II) or Cd­(II) binding perturbs the oxidative folding pathway of HD5_red to HD5_ox. Whereas HD5_red is highly susceptible to proteolytic degradation, the Zn­(II)-bound form displays resistance to hydrolytic breakdown by trypsin and other proteases. The ability of a reduced defensin peptide to coordinate Zn­(II) provides a putative mechanism for how these peptides persist <i>in vivo</i>

NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys³–Cys³¹, Cys⁵–Cys²⁰, and Cys¹⁰–Cys³⁰) in the oxidized form (HD5_ox). To probe the solution structure and oligomerization properties of HD5_ox, and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to <i>in vitro</i> peptide stability assays. The NMR solution structure of HD5_ox, solved at pH 4.0 in 90:10 H₂O/D₂O, is presented (PDB: 2LXZ). Relaxation <i>T</i>₁/<i>T</i>₂ measurements and the rotational correlation time (τ_c) estimated from a ¹⁵N-TRACT experiment demonstrate that HD5_ox is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19–21 (Val¹⁹–Cys²⁰–Glu²¹) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7–14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5_ox is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14 363 Da were determined for HD5_ox at pH 7.0, supporting a tetrameric form ([HD5_ox] ≥ 30 μM). At pH 2.0, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7079 Da, corresponding to a HD5_ox dimer, were obtained. Millimolar concentrations of NaCl, CaCl₂, and MgCl₂ have a negligible effect on the HD5_ox sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environmentally sensitive behavior of HD5_ox in solution, and provide important insights into HD5_ox structure/activity relationships and the requirements for antimicrobial action

Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus

Human α-defensin 5 (HD5, HD5[subscript ox] to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys[superscript 3]-Cys[superscript 31], Cys[superscript 5]-Cys[superscript 20], Cys[superscript 10]-Cys[superscript 30]) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser[superscript hexa]], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S–S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5[subscript ox] against this Gram-positive bacterial strain. This observation supports the notion that the HD5[subscript ox] mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem.284, 29180−29192] and that the native disulfide array is a requirement for its activity against S. aureus.Massachusetts Institute of Technology. Biophysical Instrumentation Facility ((NSF- 0070319)Massachusetts Institute of Technology. Biophysical Instrumentation Facility (NIH GM68762

NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys[superscript 3]–Cys[superscript 31], Cys[superscript 5]–Cys[superscript 20], and Cys[superscript 10]–Cys[superscript 30]) in the oxidized form (HD5[subscript ox]). To probe the solution structure and oligomerization properties of HD5[subscript ox], and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to in vitro peptide stability assays. The NMR solution structure of HD5[subscript ox], solved at pH 4.0 in 90:10 H2O/D2O, is presented (PDB: 2LXZ). Relaxation T[subscript 1]/T[subscript 2] measurements and the rotational correlation time (τc) estimated from a [superscript 15]N-TRACT experiment demonstrate that HD5[subscript ox] is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19–21 (Val[superscript 19]–Cys[superscript 20]–Glu[superscript 21]) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7–14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5[subscript ox] is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14 363 Da were determined for HD5[subscript ox] at pH 7.0, supporting a tetrameric form ([HD5ox] ≥ 30 μM). At pH 2.0, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7079 Da, corresponding to a HD5ox dimer, were obtained. Millimolar concentrations of NaCl, CaCl[subscript 2], and MgCl[subscript 2] have a negligible effect on the HD5[subscript ox] sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environmentally sensitive behavior of HD5[subscript ox] in solution, and provide important insights into HD5[subscript ox] structure/activity relationships and the requirements for antimicrobial action.National Institutes of Health (U.S.) (NIH Grant DP2OD007045)National Institute of General Medical Sciences (U.S.) (NIH Grant P01 GM047467)National Institute for Biomedical Imaging and Bioengineering (U.S.)National Institute for Biomedical Imaging and Bioengineering (U.S.) (NIH Grant EB-002026)Massachusetts Institute of Technology. Dept. of Chemistr