Glycerol monolaurate (GML) is a mild surfactant commonly used in the food and cosmetics industry as an emulsifier. Although not identified with having significant human toxicity, GML inhibits the growth of some gram-positive bacteria at a concentration of 20 μg/ml, and at more dilute concentrations it inhibits virulence factor production. Previous studies have shown inhibition of toxic shock syndrome toxin 1, alpha-hemolysin, and protein A from Staphylococcus aureus
, as well as inhibition of superantigens from Streptococcus pyogenes
). GML also inhibits the induction of vancomycin resistance in Enterococcus faecalis
). Because of the success of GML with these organisms, GML is being investigated as a possible therapeutic agent to be used in wound dressings or in tampons to prevent the toxin production by these organisms.
The mechanism of GML action on these organisms is unclear. GML contains the fatty acid lauric acid, attached to a glycerol molecule. GML has 12 carbon molecules as its fatty acid backbone chain, which makes it span exactly one-half the width of a lipid bilayer. Because of its length and lipophilic nature, GML is thought to act at the membrane of organisms by interfering with signal transduction, most likely through two-component mechanisms. Such inhibition was suggested in at least one case through studies showing that GML inhibits the induction of vancomycin resistance in E. faecalis
, which is a system that is controlled by a well-known two-component system, VanS and VanR (11
). However, GML does not affect RNA III, which acts through the well-characterized agr
two-component system in S. aureus
that controls expression of secreted virulence factors (9
). This leaves the possibility that there are yet-uncharacterized two-component systems through which GML may be acting.
Because of GML effects on other gram-positive organisms, we undertook experiments to determine the effect GML on Bacillus anthracis. B. anthracis, the causative agent of anthrax, is a spore-forming organism whose natural environment is the soil. Although an important agricultural risk, anthrax is currently in the limelight, being ranked as a top risk as an agent of biological terrorism. Therefore, it is important to understand mechanisms that control the expression of virulence factors as well to develop potential treatments in order to be well prepared for sudden outbreaks of the disease.
Anthrax has three proteins that make up two anthrax toxins, lethal factor (LF) and edema factor (EF). The toxin components consist of protective antigen (PA), LF, and EF. Their genes, pagA encoding PA, lef encoding LF, and cya encoding EF, are located on pXO1, one of two plasmids in B. anthracis that are essential for virulence. These toxin proteins are unique in that they are not active by themselves. Both LF and EF must be coupled to PA to form the active toxins lethal toxin and edema toxin. The other plasmid, pXO2, carries the genes capABC, coding for an antiphagocytic capsule.
Environmental factors are extremely important for the expression of toxin genes, most notably temperature and a CO2
). The toxin genes are controlled by a regulator, atxA
, also located on the pXO1 plasmid, as well as abrB
, a chromosomal gene, inhibiting toxin production until post-exponential phase (2
). Yet, there is much unknown about toxin regulation and under what roles certain genes play in regulation. For example, although the presence of atxA
is essential for anthrax toxin expression, modified expression levels of atxA
have yet to be matched with a changing CO2
In the present studies, GML was added to broth cultures, and growth and toxin production were analyzed. It was found that GML concentrations greater than 10 μg/ml had growth-inhibitory effects on B. anthracis. However, a 10-μg/ml concentration of GML inhibited the virulence factors PA and LF. Through quantitative reverse transcription-PCR (qRT-PCR), it was determined that the inhibition was at the transcriptional level, supporting other evidence that GML acts via signal transduction. atxA was found to be up-regulated in the presence of GML. These studies provide insights into toxin gene expression as well as treatments for anthrax that may be useful as adjunct therapies with antibiotics.