Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

Rodríguez-Rubio, Lorena; Gutiérrez, Dolores; Martínez, Beatriz; Rodrı́guez, Ana; Garcı́a, Pilar

doi:10.1128/aem.00018-12

Cited by 21 publications

(16 citation statements)

References 40 publications

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“…Although this confirms the presence of AMPs in the supernatants, it illustrates that a significant amount of peptide still remains inside the cells. Similar observations have also been observed with other recombinant peptides fused to the Usp45 signal peptide 47 . The protein size and the particular combination of signal peptide and mature protein are factors that may be limiting the secretion of the peptides.…”

Section: Resultssupporting

confidence: 87%

Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

et al. 2013

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We present results of tests with recombinant Lactococcus lactis that produce and secrete heterologous antimicrobial peptides with activity against Gram-negative pathogenic Escherichia coli and Salmonella. In an initial screening, the activities of numerous candidate antimicrobial peptides, made by solid state synthesis, were assessed against several indicator pathogenic E. coli and Salmonella strains. Peptides A3APO and Alyteserin were selected as top performers based on high antimicrobial activity against the pathogens tested and on significantly lower antimicrobial activity against L. lactis. Expression cassettes containing the signal peptide of the protein Usp45 fused to the codon optimized sequence of mature A3APO and Alyteserin were cloned under the control of a nisin-inducible promoter nisA and transformed into L. lactis IL1403. The resulting recombinant strains were induced to express and secrete both peptides. A3APO- and Alyteserin-containing supernatants from these recombinant L. lactis inhibited the growth of pathogenic E. coli and Salmonella by up to 20-fold, while maintaining the host’s viability. This system may serve as a model for the production and delivery of antimicrobial peptides by lactic acid bacteria to target Gram-negative pathogenic bacteria populations.

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Section: Resultssupporting

confidence: 87%

Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

et al. 2013

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“…A phage-associated lysin could rupture dead and living S. aureus cells [33]. In addition, phage lytic enzymes from staphylococcal phages 80a [3], Twort [14], 187 [13], phi11 [22], vB-SauS-phi-IPLA88 [28,29], and K [26] have been described. The in vitro lytic activity of LysK enzyme was found to be effective against several staphylococcal species including methicillin-resistant S. aureus strains [22].…”

Section: Introductionmentioning

confidence: 99%

Expression and lytic efficacy assessment of theStaphylococcus aureusphage SA4 lysin gene

et al. 2013

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Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.

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“…; Rodriguez‐Rubio et al . ; Walmagh et al . ) and endolysins with activity against pathogens of medical and economic concern (Son et al .…”

Section: Resultsmentioning

confidence: 99%

“…In similar combinatorial studies, LysH5 endolysin was combined with the secretion signal sequence of bacteriocin Lcn972 to increase extracellular excretion of active endolysin (Rodriguez‐Rubio et al . ), while the streptococcal phage lysin endopeptidase domain was combined with the cell wall‐binding domains of the staphylococcal phage lysin LysK and lysostaphin, resulting in killing activity against Staphylococcus aureus isolates from livestock (Schmelcher et al . ).…”

Section: Resultsmentioning

confidence: 99%

“…The susceptible genera contain pathogenic bacteria of medical and economic importance. Recent studies characterizing new endolysins (Son et al 2010a;Rodriguez-Rubio et al 2012;Walmagh et al 2012) and endolysins with activity against pathogens of medical and economic concern (Son et al preservatives (Settanni and Corsetti 2008;Khan et al 2010), agricultural livestock antimicrobials (Joerger 2003), biosensors for detection of bacterial cells (Kretzer et al 2007;Tolba et al 2012), and inhibitors of biofilm (Son et al 2010b). Mitrecin A is organized in a modular arrangement of domains with an N-terminal cell-binding domain and a C-terminal catalytic domain, a common feature in endolysins of Gram-positive bacteria (Garcia et al 1988) and can be exploited for the development of designed functions.…”

Section: Resultsmentioning

confidence: 99%

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Mitrecin A , an endolysin‐like bacteriolytic enzyme from a newly isolated soil streptomycete

Farris

Steinberg

2014

Lett Appl Microbiol

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An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14·3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia.Significance and Impact of the StudyThe gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

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Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

Cited by 21 publications

References 40 publications

Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

Expression and lytic efficacy assessment of theStaphylococcus aureusphage SA4 lysin gene

Mitrecin A , an endolysin‐like bacteriolytic enzyme from a newly isolated soil streptomycete

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