Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins

The immunity proteins of pediocin-like bacteriocins show a high degree of specificity with respect to the pediocin-like bacteriocin they recognize and confer immunity to. The aim of this study was to identify regions of the immunity proteins that are involved in this specific recognition. Six different hybrid immunity proteins were constructed from three different pediocin-like bacteriocin immunity proteins that have similar sequences but confer resistance to different bacteriocins. These hybrid immunity proteins were then tested for their ability to confer immunity to various pediocin-like bacteriocins. The specificities of the hybrid immunity proteins proved to be similar to those of the immunity proteins from which the C-terminal halves were derived, thus revealing that the C-terminal half of immunity proteins for pediocin-like bacteriocins contains a domain that is involved in specific recognition of the bacteriocins they confer immunity to. Moreover, the results also revealed that the effectiveness of an immunity protein is strain dependent and that its functionality thus depends in part on interplay with strain-dependent factors. To further investigate the structure-function relationship of these immunity proteins, the enterocin A and leucocin A immunity proteins (EntA-im and LeuA-im) were purified to homogeneity and structurally analyzed under various conditions by Circular dichroism (CD) spectroscopy. The results revealed that both immunity proteins are α-helical and well structured in an aqueous environment, the denaturing temperature being 78.5°C for EntA-im and 58.0°C for LeuA-im. The CD spectra also revealed that there was no further increase in the structuring or α-helical content when the immunity proteins were exposed to dodecylphosphocholine micelles or dioleoyl-l-α-phosphatidyl-dl-glycerol (DOPG) liposomes, indicating that the immunity proteins, in contrast to the bacteriocins, do not interact extensively with membranes. They may nevertheless be loosely associated with the membrane, possibly as peripheral membrane proteins, thus enabling them to interact with their cognate bacteriocin

Extracellular Identification of a Processed Type II ComR/ComS Pheromone of Streptococcus mutans

The competence-stimulating peptide (CSP) and the sigX-inducing peptide (XIP) are known to induce Streptococcus mutans competence for genetic transformation. For both pheromones, direct identification of the native peptides has not been accomplished. The fact that extracellular XIP activity was recently observed in a chemically defined medium devoid of peptides, as mentioned in an accompanying paper (K. Desai, L. Mashburn-Warren, M. J. Federle, and D. A. Morrison, J. Bacteriol. 194:3774–3780, 2012), provided ideal conditions for native XIP identification. To search for the XIP identity, culture supernatants were filtered to select for peptides of less than 3 kDa, followed by C18 extraction. One peptide, not detected in the supernatant of a comS deletion mutant, was identified by tandem mass spectrometry (MS/MS) fragmentation as identical to the ComS C-terminal sequence GLDWWSL. ComS processing did not require Eep, a peptidase involved in processing or import of bacterial small hydrophobic peptides, since eep deletion had no inhibitory effect on XIP production or on synthetic XIP response. We investigated whether extracellular CSP was also produced. A reporter assay for CSP activity detection, as well as MS analysis of supernatants, revealed that CSP was not present at detectable levels. In addition, a mutant with deletion of the CSP-encoding gene comC produced endogenous XIP levels similar to those of a nondeletion mutant. The results indicate that XIP pheromone production is a natural phenomenon that may occur in the absence of natural CSP pheromone activity and that the heptapeptide GLDWWSL is an extracellular processed form of ComS, possibly the active XIP pheromone. This is the first report of direct identification of a ComR/ComS pheromone

Enhanced neural drive after maximal strength training in multiple sclerosis patients

Multiple sclerosis (MS) patients suffer from impaired muscle activation and lower limb strength. Strength training enhances muscle activation and muscle strength, but neural adaptations to strength training remain unexplored in MS patients. The hypothesis was that maximal strength training (MST) using high loads and few repetitions would improve central neural drive and thus strength capacity of MS patients. 14 MS patients staying at a national MS rehabilitation center were randomly assigned to a MST group or a control group (CG). Both groups received “today’s treatment”. In addition, the MST group trained 4 × 4 repetitions of unilateral dynamic leg press and plantar flexion 5 days a week for 3 weeks. Neural adaptations of the soleus muscle were assessed by surface electromyography (EMG) activity, and by superimposed H-reflexes and V-waves obtained during maximum voluntary isometric plantar flexor contractions (MVCs). H-reflexes and V-waves were normalized by the M-wave (H SUP/M SUP, V/M SUP, respectively). In the MST group, MVC increased by 20 ± 9% (P < 0.05). Soleus EMG activity and V/M SUP ratio increased by 40 and 55%, respectively, in the MST group compared to the CG (P ≤ 0.05). The H SUP/M SUP ratio remained unchanged. No change was apparent in the CG. MST group subjects were able to complete all training sessions. No adverse effects were reported. This randomized study provides evidence that MST is effective of augmenting the magnitude of efferent motor output of spinal motor neurons in MS patients, alleviating some neuromuscular symptoms linked to the disease

Analysis of the Two-Peptide Bacteriocins Lactococcin G and Enterocin 1071 by Site-Directed Mutagenesis▿

The two peptides (Lcn-α and Lcn-β) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-α and Ent-β) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-α+Ent-β had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-α+Lcn-β), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-α+Lcn-β) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-α+Ent-β), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-α is more active against lactococci in combination with Lcn-β and more active against enterococci in combination with Ent-β suggests that the β peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the β peptide seem to be important for specificity, since Ent-α combined with an Ent-β variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-α+Ent-β. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-β had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the α peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-α influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2- to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only ∼2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-α and Lcn-β, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an ∼10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-α and Lcn-β, respectively). Interestingly, the Lcn-to-Ent F19L+G20A mutation in an Lcn-Ent-β hybrid peptide was more detrimental when the altered peptide was combined with Lcn-α (>10-fold reduction) than when it was combined with Ent-α (∼2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif) in the β peptide may be involved in a specific interaction with the cognate α peptide. It is also noteworthy that the K2P and A7P mutations in Lcn-β reduced the activity only ∼2-fold, suggesting that the first seven residues in the β peptides do not form an α-helix

The Continuing Story of Class IIa Bacteriocins

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against

Functional maximal strength training induces neural transfer to single-joint tasks

The purpose of this study was to investigate whether neural adaptations following functional multiplejoint leg press training can induce neural adaptations to the plantar Xexor muscles in a single-joint contraction task. Subjects were randomised to a maximal strength training (MST) (n = 10) or a control group (n = 9). MST consisted of 24 sessions (8 weeks) of 4 x 4 repetitions of horizontal leg press using maximal intended velocity in the concentric phase with the movement ending in a plantar Xexion. Neural adaptations in the soleus and gastrocnemius medialis (GM) were assessed by surface electromyographic activity and V-waves during maximum voluntary isometric contraction (MVIC), and also by H-reXexes in the soleus during rest and 20% MVIC. One repetition maximum leg press increased by 44 ± 14% (mean ± SD; P < 0.01). Plantar Xexion MVIC increased by 20 ± 14% (P < 0.01), accompanied by 13 ± 19% (P < 0.05) increase in soleus, but not GM surface electromyography. Soleus V/MSUP increased by 53 ± 66% and in GM by 59 ± 64% (P < 0.05). Normalised soleus H-reXexes remained unchanged by training. No changes occurred in the control group. These results suggest that leg press MST can induce neural adaptations in a single-joint plantar Xexion MVIC task