368
15.2.2 Purification of Bacteriocin E50-52A
Sosunov et al. (2007) isolated E50–52A from E. faecalis by growing the bacteria in
modified Kugler broth media. The protein was precipitated with ammonium sulfate
from centrifuged culture supernatant. After dialysis, this crude antimicrobial preparation (CAP) was subjected to gel filtration on Superose 12HR 16/50 columns
(Pharmacia, Uppsala, Sweden), and eluted fractions were tested for antimicrobial
activity against Campylobacter jejuni strains. The bioactive fractions were purified
on 20 cm Mono Q HR 5/5 columns and on 20 cm CM-Sepharose columns
(Pharmacia). The molecular weights of bioactive fractions were determined by electrophoresis and confirmed by matrix-assisted laser-desorption ionization–time-offlight (MALDI–TOF) mass spectrometry. Edman degradation was used to determine
the amino acid sequence of the peptide, and a highly conserved motif “YGNGV”
was found to be present in all class IIa bacteriocins as the amino-terminal sequence.
15.2.3 Ub2
Ub2 is a peptide derived from ubiquitin identified in lysosome (Kieffer et al. 2003).
Immune response kills mycobacteria by trafficking of the bacterium to the lysosome – a phenomenon called autophagy. Alonso et al. (2007) found that solubilized
lysosomes isolated from resting bone marrow–derived macrophages were bactericidal toward both M. tuberculosis and M. smegmatis. These solubilized lysosomal
materials were fractionated by HPLC. The bioactive fraction on analysis by mass
spectrometry revealed the bactericidal activity to be associated with ubiquitin (Ub)
peptides. Purified ubiquitin lacked bactericidal activity, but on digestion with
cathepsin protease, they found few peptides that exhibited bactericidal activity.
Similarly, they found the de novo synthesized peptide Ub2 with amino acid sequence
STLHLVLRLRGG to be bactericidal against mycobacterial species (Foss et al.
2012). The bactericidal activity of ubiquitin was confirmed by Purdy et al. (2009).
When lysosomal extract was treated with polyclonal antibodies against ubiquitin,
immunodepletion of ubiquitin led to diminished activity of the extract. Since the
lysosomal lysates from autophagic and activated macrophages have increased levels
of ubiquitin, these macrophages exhibit increased microbicidal activity when compared to resting macrophages. Further bactericidal experiments by Purdy et al.
(2009) using synthesized Ub2 at different pH against mycobacterial cultures grown
in 7H9 media confirmed that Ub2 exhibited maximum bactericidal activity between
pH 5.5 and 6. These experiments further confirm the effective role of Ub peptides at
relevant physiological pH, as the internal pH of phagosomes varies from 5.5 to 6.2
depending on its maturity. The β-sheet in Ub2 is primarily responsible for the membrane targeting of the molecule (Foss et al. 2012).
S.M. Thayil and A.K. Kesavan
15.2.2 Purification of Bacteriocin E50-52A
Sosunov et al. (2007) isolated E50–52A from E. faecalis by growing the bacteria in
modified Kugler broth media. The protein was precipitated with ammonium sulfate
from centrifuged culture supernatant. After dialysis, this crude antimicrobial preparation (CAP) was subjected to gel filtration on Superose 12HR 16/50 columns
(Pharmacia, Uppsala, Sweden), and eluted fractions were tested for antimicrobial
activity against Campylobacter jejuni strains. The bioactive fractions were purified
on 20 cm Mono Q HR 5/5 columns and on 20 cm CM-Sepharose columns
(Pharmacia). The molecular weights of bioactive fractions were determined by electrophoresis and confirmed by matrix-assisted laser-desorption ionization–time-offlight (MALDI–TOF) mass spectrometry. Edman degradation was used to determine
the amino acid sequence of the peptide, and a highly conserved motif “YGNGV”
was found to be present in all class IIa bacteriocins as the amino-terminal sequence.
15.2.3 Ub2
Ub2 is a peptide derived from ubiquitin identified in lysosome (Kieffer et al. 2003).
Immune response kills mycobacteria by trafficking of the bacterium to the lysosome – a phenomenon called autophagy. Alonso et al. (2007) found that solubilized
lysosomes isolated from resting bone marrow–derived macrophages were bactericidal toward both M. tuberculosis and M. smegmatis. These solubilized lysosomal
materials were fractionated by HPLC. The bioactive fraction on analysis by mass
spectrometry revealed the bactericidal activity to be associated with ubiquitin (Ub)
peptides. Purified ubiquitin lacked bactericidal activity, but on digestion with
cathepsin protease, they found few peptides that exhibited bactericidal activity.
Similarly, they found the de novo synthesized peptide Ub2 with amino acid sequence
STLHLVLRLRGG to be bactericidal against mycobacterial species (Foss et al.
2012). The bactericidal activity of ubiquitin was confirmed by Purdy et al. (2009).
When lysosomal extract was treated with polyclonal antibodies against ubiquitin,
immunodepletion of ubiquitin led to diminished activity of the extract. Since the
lysosomal lysates from autophagic and activated macrophages have increased levels
of ubiquitin, these macrophages exhibit increased microbicidal activity when compared to resting macrophages. Further bactericidal experiments by Purdy et al.
(2009) using synthesized Ub2 at different pH against mycobacterial cultures grown
in 7H9 media confirmed that Ub2 exhibited maximum bactericidal activity between
pH 5.5 and 6. These experiments further confirm the effective role of Ub peptides at
relevant physiological pH, as the internal pH of phagosomes varies from 5.5 to 6.2
depending on its maturity. The β-sheet in Ub2 is primarily responsible for the membrane targeting of the molecule (Foss et al. 2012).
S.M. Thayil and A.K. Kesavan