372
15.2.9 Preparation of Lacticin 3147
McAuliffe et al. (1998) purified the bacteriocin by growing L. lactis subsp. lactis
DPC3147 in tryptone–yeast broth. The culture was centrifuged, and culture supernatant was passed through XAD-16 bead. The bioactive bacteriocin was applied to
activate C18 reverse-phase column. The column was washed with 30% ethanol, and
the active bacteriocin was eluted in 70% isopropanol–10 mM acetic acid (pH 2).
Active fractions thus obtained were concentrated and subjected to a fast protein
liquid chromatography (FPLC). Extracellular ATP levels, transmembrane electric
potentials, and intra- and extracellular K
+
concentration were used to determine the
bacteriocin potential.
15.2.10 LLKK and II-D
Two unnatural d-amino acid synthetic peptides, LLKK and II-D, have shown promising results against both MDR and susceptible clinical isolates in vitro within 4
days of treatment. The peptides were resistant to proteases. Live-cell imaging and
confocal microscopy revealed that the peptide could transverse the mycobacterial
membrane and accumulate in the cytoplasm (Khara et al. 2016).
15.3 LPS Inhibitors
15.3.1 LL37
LL37 is the C-terminal domain of human cathelicidin known as human cationic
antimicrobial protein (hCAP18) (Mendez-Samperio 2010). Human cathelicidin
exhibits various functions like immunomodulation and chemotaxic activity in addition to microbial inhibition (Bowdish et al. 2006). Cathelicidin-mediated antimycobacterial effect of vitamin D was demonstrated by Liu et al. (2007). In this
experiment, the human monocytic cell line THP1 cell was infected with
Mycobacterium H37Ra and stimulated with 1,25-dihydroxyvitamin D3. The inhibition of mycobacterial growth was recorded in the presence of cathelicidins; upon
knocking down cathelicidin production using siRNA, the antimycobacterial effect
of vitamin D was lost. This led to the conclusion that the induction and expression
of cathelicidins were important for the antimycobacterial effect of vitamin D
ex vivo. Martineau et al. (2007) observed an iron-dependent antimycobacterial
effect of LL37. On prolonged exposure of LL37 (5 g/mL), they found a 1.8-fold
reduction in mycobacterial colony forming units (CFU) in iron-depleted cultures
when compared to cultures that were not exposed to LL37. The antimicrobial
potency of LL37 peptide was increased by substituting Q22, N30 and D26 residues
with positively charged lysine residues forming LLKKK18 peptide. Silver nanoparticles coated with LLKKK18 have shown promising antimycobacterial activity.
Recently, the use of self-assembling hyaluronic acid (HA) nanogels for the delivery
S.M. Thayil and A.K. Kesavan
15.2.9 Preparation of Lacticin 3147
McAuliffe et al. (1998) purified the bacteriocin by growing L. lactis subsp. lactis
DPC3147 in tryptone–yeast broth. The culture was centrifuged, and culture supernatant was passed through XAD-16 bead. The bioactive bacteriocin was applied to
activate C18 reverse-phase column. The column was washed with 30% ethanol, and
the active bacteriocin was eluted in 70% isopropanol–10 mM acetic acid (pH 2).
Active fractions thus obtained were concentrated and subjected to a fast protein
liquid chromatography (FPLC). Extracellular ATP levels, transmembrane electric
potentials, and intra- and extracellular K
+
concentration were used to determine the
bacteriocin potential.
15.2.10 LLKK and II-D
Two unnatural d-amino acid synthetic peptides, LLKK and II-D, have shown promising results against both MDR and susceptible clinical isolates in vitro within 4
days of treatment. The peptides were resistant to proteases. Live-cell imaging and
confocal microscopy revealed that the peptide could transverse the mycobacterial
membrane and accumulate in the cytoplasm (Khara et al. 2016).
15.3 LPS Inhibitors
15.3.1 LL37
LL37 is the C-terminal domain of human cathelicidin known as human cationic
antimicrobial protein (hCAP18) (Mendez-Samperio 2010). Human cathelicidin
exhibits various functions like immunomodulation and chemotaxic activity in addition to microbial inhibition (Bowdish et al. 2006). Cathelicidin-mediated antimycobacterial effect of vitamin D was demonstrated by Liu et al. (2007). In this
experiment, the human monocytic cell line THP1 cell was infected with
Mycobacterium H37Ra and stimulated with 1,25-dihydroxyvitamin D3. The inhibition of mycobacterial growth was recorded in the presence of cathelicidins; upon
knocking down cathelicidin production using siRNA, the antimycobacterial effect
of vitamin D was lost. This led to the conclusion that the induction and expression
of cathelicidins were important for the antimycobacterial effect of vitamin D
ex vivo. Martineau et al. (2007) observed an iron-dependent antimycobacterial
effect of LL37. On prolonged exposure of LL37 (5 g/mL), they found a 1.8-fold
reduction in mycobacterial colony forming units (CFU) in iron-depleted cultures
when compared to cultures that were not exposed to LL37. The antimicrobial
potency of LL37 peptide was increased by substituting Q22, N30 and D26 residues
with positively charged lysine residues forming LLKKK18 peptide. Silver nanoparticles coated with LLKKK18 have shown promising antimycobacterial activity.
Recently, the use of self-assembling hyaluronic acid (HA) nanogels for the delivery
S.M. Thayil and A.K. Kesavan