Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

peer-reviewedIt is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.This work was supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H. and R.P.R. with SFI Principal Investigator funding

A Specialized Polythioamide‐Binding Protein Confers Antibiotic Self‐Resistance in Anaerobic Bacteria

Understanding antibiotic resistance mechanisms is central to the development of anti‐infective therapies and genomics‐based drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from less‐explored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers self‐resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum . Bioinformatics, biochemical analyses, and X‐ray crystallography revealed CtaZ as a founding member of a new group of GyrI‐like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand‐optimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrI‐like domain in self‐resistance and unprecedented thioamide binding, this work aids in uncovering related drug‐resistance mechanisms

The surface-associated exopolysaccharide of Bifidobacterium longum 35624 plays an essential role in dampening host proinflammatory responses and repressing local TH17 responses

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro. In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses

'Bac' to the future: bioengineering lantibiotics for designer purposes

Abstract Bacteriocins are bacterially produced peptides or proteins that inhibit the growth of other bacterial strains. They can have a broad (effective against multiple genera) or narrow (effective against specific species) spectrum of activity. The diversity of bacteriocins found in Nature, in terms of both spectrum of activity and physiochemical properties, offers the possibility of multiple applications in the food and pharmaceutical industries. However, traditional screening strategies may not provide a sufficient range of natural molecules with specifically desired properties. Research suggests that bioengineering of existing inhibitors has the potential to address this issue, extending the application of natural bacteriocins for use in novel settings and against different targets. In the present paper, we discuss the successful implementation of bioengineering strategies to alter and even improve the functional characteristics of a bacteriocin, using the prototypical lantibiotic nisin as an example. Additionally, we describe the recent use of the nisin-modification machinery in vivo to enhance the properties of medically significant peptides

Determination of the extent to which supervisors, head nurses, and staff nurses in general hospitals have specific understanding of range of joint motion

Thesis (M.S.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.2031-01-0

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.This work was supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H. and R.P.R. with SFI Principal Investigator funding

Data from: Identification of the Minimal Cytolytic Unit for Streptolysin S and an Expansion of the Toxin Family

Background: Streptolysin S (SLS) is a cytolytic virulence factor produced by the human pathogen Streptococcus pyogenes and other Streptococcus species. Related “SLS-like” toxins have been characterized in select strains of Clostridium and Listeria, with homologous clusters bioinformatically identified in a variety of other species. SLS is a member of the thiazole/oxazole-modified microcin (TOMM) family of natural products. The structure of SLS has yet to be deciphered and many questions remain regarding its structure-activity relationships. Results: In this work, we assessed the hemolytic activity of a series of C-terminally truncated SLS peptides expressed in SLS-deficient S. pyogenes. Our data indicate that while the N-terminal poly-heterocyclizable (NPH) region of SLS substantially contributes to its bioactivity, the variable C-terminal region of the toxin is largely dispensable. Through genome mining we identified additional SLS-like clusters in diverse Firmicutes, Spirochaetes and Actinobacteria. Among the Spirochaete clusters, naturally truncated SLS-like precursors were found in the genomes of three Lyme disease-causing Borrelia burgdorferi sensu lato (Bbsl) strains. Although unable to restore hemolysis in SLS-deficient S. pyogenes, a Bbsl SLS-like precursor peptide was converted to a cytolysin using purified SLS biosynthetic enzymes. A PCR-based screen demonstrated that SLS-like clusters are substantially more prevalent in Bbsl than inferred from publicly available genome sequences. Conclusions: The mutagenesis data described herein allowed us to define the minimal cytolytic unit of SLS as the NPH region. Interestingly, this region is found in all characterized TOMM cytolysins, as well as the novel putative TOMM cytolysins we discovered. We propose that this conserved region represents the defining feature of the SLS-like TOMM family. We demonstrate the cytolytic potential of a Bbsl SLS-like precursor peptide, which is of similar length to the SLS minimal cytolytic unit, when modified with purified SLS biosynthetic enzymes. As such, we speculate that some Borrelia have the potential to produce a TOMM cytolysin, although the biological significance of this finding remains to be determined. In addition to providing new insight into the structure-activity relationships of SLS, this study greatly expands the cytolysin group of TOMMs

The surface-associated exopolysaccharide of bifidobacterium longum 35624 plays an essential role in dampening host proinflammatory responses and repressing local TH17 responses

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses

Mass Spectrometry Analysis and Bioactivity Determination of Nisin K12X Bank.

<p>Observed molecular mass (+/−0.25 Da) from MALDI-TOF MS analysis of NZ9800 pDF05-K12X producers. ND: not detected. * represents dehydrated forms (hydrophobic modified residues), i.e. Dhb in the case of T and Dha in the case of S.</p><p>Bioactivity of NZ9800 pDF05-K12X producers against <i>L. lactis</i> HP. Values given are the mean of triplicate deferred antagonism assays and represent zone of inhibition (diameter of zone minus diameter of bacterial growth) expressed as a percentage compared to that of the wild-type nisin producer at 100%. S.D.: Standard Deviation; Relative Standard Deviation <10% for each given value. N/A: not applicable. All values in bold reached statistical significance compared to nisin control (K) (Student's t-test: P<0.05).</p