46 4 ART for Antibacterial Infection
5.2.3 Discussion
The nonpathogenic B. subtilis and pathogenic B. anthracis as well as other
G+ bacteria generate NO by their own bNOS that contains a prosthetic heme moi-
ety for the reduction of a catalytic center from [Fe^3 +] to [Fe^2 +] (Gusarov et al.
2008 ). Our present research showed that B. licheniformis can accumulate nitrate
and nitrite, the oxidized products of NO, in the media under oxidative stress
conditions including hypoxia and hypoxia + cold. This finding is supported by
a previous report describing that NO is not consumed and can accumulate in
the microenvironment of human tissue at lower O 2 concentrations (Taylor and
Moncada 2010 ). Furthermore, we also found that bacterial NO production can be
repressed by ART or l-NMMA, suggesting that bNOS is involved in the oxidative
stress-induced NO production.
Although E. coli strains generating NO are independent on bNOS, they have
an enzyme complex encompassing nitrite reductase, flavohemoglobin, and
NO-sensing regulator responsible for NO production (Corker and Poole 2003 ).
Indeed, we observed an elevation of NO levels in E. coli overnight cultures. The
NO level, either in the presence or absence of ART or ART + CEF, was detect-
able, but relatively low (below 12 μM), much lower than that in B. licheniformis
(80–120 μM), suggesting that such a low NO level in E. coli is insufficient to pro-
tect bacteria from antibiotic attacking. ART may be directly cytotoxic to bacteria
because ART can be converted to carbon-centered free radicals in vivo. The inhibi-
tion of a bacterial multidrug efflux pump system, of course, should represent an
alternative mechanism of ART sensitizing antibiotics in E. coli (Li et al. 2011 ).
Given that heme alkylation by ART has been verified by identifying the ART-
heme adducts in malaria-infected mice (Robert et al. 2005 ), and considering that
an ART-heme interaction has been also confirmed in tumor cells (Zhang and
Gerhard 2009 ), we proposed that ART should also interact with bacterial hemo-
proteins. In addition to bNOS, CAT is also a hemoprotein in Bacilus bacteria (Bol
and Yasbin 1994 ). In the present study, we did detect the presence of ART-heme
conjugates in B. licheniformis after incubation with ART for three hours. Later on,
however, ART-heme conjugates cannot be detected, probably because of the deg-
radation of conjugates following bacterial death. This situation is similar with our
observation in tumor cells (Zeng and Zhang 2011 ).
Until recently, there has no literature describing the impact of ART on bacterial
CAT. Actually, our data showed that CAT activity is considerably reduced once ART
was included. For example, a higher CAT activity (25 units/ml) for 24 h-cultures was
measured in 25 μg/ml RIF, but it is sharply declined to below two units/ml when
25 μg/ml RIF was combined with 60 μg/ml ART. Mechanistically, CAT is believed
to be activated by NO in bacteria through diminishing the rate of cystine reduction to
cysteine, which drives the Fenton reaction and simultaneously inhibits CAT activity
(Gusarov and Nudler 2005 ). Due to the CAT inhibition, hydroxyl radicals derived
from an excess H 2 O 2 would exhibit an extreme toxicity to bacterial DNA through
base modifications and strand breaks (Woodmansee and Imlay 2002 ).