369
15.2.4 Granulysin
Granulysin is a cationic protein and a member of the saponin-like protein family
(Pena and Krensky 1997; Pitabut et al. 2013). Granulysin has an important role in
cytotoxic T cell response to intracellular bacterial pathogens (Mueller et al. 2011).
The effect of granulysin on the viability of virulent M. tuberculosis was examined
by culturing M. tuberculosis in 7H9 media in the presence of various concentrations
of granulysin (Stenger et al. 1998). These experiments showed that the granulysin
killed 90% of M. tuberculosis in vitro within 72 h in a dose-dependent manner.
However, this antimycobacterial activity was not detected ex vivo, when granulysin
was added to M. tuberculosis–infected macrophages.
Okada et al. (2011) have shown promising results against both MDR-TB and
XDR-TB in mice and a monkey model of infection using a DNA vaccine expressing
mycobacterial heat shock protein 65 and IL-12 as well as a granulysin vaccine.
Granulysin-derived synthetic peptides GranF2 and G13 have been shown to inhibit
in vitro growth of clinical isolates of MDR tuberculosis strains. Toro et al. (2006)
have demonstrated that GranF2 (Andreu et al. 1999) and G13 (Wang et al. 2000)
inhibited multidrug-resistant M. tuberculosis to a higher extent when compared to
inhibition of drug-susceptible strains. GranF2 in combination with first-line drug
ethambutol enhanced its antibacterial effect. In combination with streptolysin O,
GranF2 efficiently reduced the intracellular growth of multidrug-resistant M. tuberculosis. The difference in the susceptibility of drug-resistant versus sensitive strain
of MTB was attributed to the difference in cell wall architecture. The drug-sensitive
strains exhibit a robust cell wall and membrane and the resistant strains have reduced
fitness and growth. Andreu et al. (1999) identified a 22-residue alpha helical
sequence and a loop to be the site with antimycobacterial activity in NK-lysin and
granulysin.
15.2.5 HNP-1
Human neutrophil peptides 1, 2 and 3 collectively called “defensins” were first purified by Ganz et al. (1985) from neutrophils and exhibited a broad range of antimicrobial activity (Liu and Modlin 2008). Sharma et al. (2000) have shown an
inhibition of M. tuberculosis H37Rv growth both in vitro and ex vivo in the presence of HNP-1. During mycobacterial infection, HNP-1 modulates the innate
response by mediating the secretion of TNF alpha required for monocyte migration
to the site of infection. Purification of human defensins HNP-1, HNP-2 and HNP-3
were done from polymorphonuclear leukocyte (PMN) cells by homogenization and
differential centrifugation. This homogenate was subjected to acetic acid extraction
to obtain the granule protein, which was fractionated by gel filtration using Bio-Gel
15 Antimycobacterial Peptides
15.2.4 Granulysin
Granulysin is a cationic protein and a member of the saponin-like protein family
(Pena and Krensky 1997; Pitabut et al. 2013). Granulysin has an important role in
cytotoxic T cell response to intracellular bacterial pathogens (Mueller et al. 2011).
The effect of granulysin on the viability of virulent M. tuberculosis was examined
by culturing M. tuberculosis in 7H9 media in the presence of various concentrations
of granulysin (Stenger et al. 1998). These experiments showed that the granulysin
killed 90% of M. tuberculosis in vitro within 72 h in a dose-dependent manner.
However, this antimycobacterial activity was not detected ex vivo, when granulysin
was added to M. tuberculosis–infected macrophages.
Okada et al. (2011) have shown promising results against both MDR-TB and
XDR-TB in mice and a monkey model of infection using a DNA vaccine expressing
mycobacterial heat shock protein 65 and IL-12 as well as a granulysin vaccine.
Granulysin-derived synthetic peptides GranF2 and G13 have been shown to inhibit
in vitro growth of clinical isolates of MDR tuberculosis strains. Toro et al. (2006)
have demonstrated that GranF2 (Andreu et al. 1999) and G13 (Wang et al. 2000)
inhibited multidrug-resistant M. tuberculosis to a higher extent when compared to
inhibition of drug-susceptible strains. GranF2 in combination with first-line drug
ethambutol enhanced its antibacterial effect. In combination with streptolysin O,
GranF2 efficiently reduced the intracellular growth of multidrug-resistant M. tuberculosis. The difference in the susceptibility of drug-resistant versus sensitive strain
of MTB was attributed to the difference in cell wall architecture. The drug-sensitive
strains exhibit a robust cell wall and membrane and the resistant strains have reduced
fitness and growth. Andreu et al. (1999) identified a 22-residue alpha helical
sequence and a loop to be the site with antimycobacterial activity in NK-lysin and
granulysin.
15.2.5 HNP-1
Human neutrophil peptides 1, 2 and 3 collectively called “defensins” were first purified by Ganz et al. (1985) from neutrophils and exhibited a broad range of antimicrobial activity (Liu and Modlin 2008). Sharma et al. (2000) have shown an
inhibition of M. tuberculosis H37Rv growth both in vitro and ex vivo in the presence of HNP-1. During mycobacterial infection, HNP-1 modulates the innate
response by mediating the secretion of TNF alpha required for monocyte migration
to the site of infection. Purification of human defensins HNP-1, HNP-2 and HNP-3
were done from polymorphonuclear leukocyte (PMN) cells by homogenization and
differential centrifugation. This homogenate was subjected to acetic acid extraction
to obtain the granule protein, which was fractionated by gel filtration using Bio-Gel
15 Antimycobacterial Peptides