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Antimicrobial Agents and Chemotherapy, June 2002, p. 1971-1973, Vol. 46, No. 6
0066-4804/02/$04.00+0 DOI: 10.1128/AAC;46.6.1971-1973.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Reducing Mortality in Salmonella enterica Serovar Typhimurium-Infected Mice with a Tripeptidic Serum Fraction
Todd A. Parker,1 Kenneth O. Willeford,1* Suzanne Parker,2 and Karyl Buddington3Department of Biochemistry and Molecular Biology,1 Department of Agricultural and Biological Engineering,2 Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi 397623
Received 11 June 2001/ Returned for modification 25 November 2001/ Accepted 6 February 2002
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Salmonellosis-induced mortality in female Swiss Webster mice decreased significantly when tripeptidic immunostimulant (TPI) was administered prophylactically. Prophylactic benefits developed in a dose-dependent manner wherein 15 mg of TPI given 1 day before challenge reduced mortality by 70%.
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Caprine serum was fractionated to isolate its complement of peptides. An isolate containing three peptides, tripeptidic immunostimulant (TPI), proved to have no bactericidal activity but significantly improved the rate of survival of chickens infected with Pasteurella multocida (9). In this study, we developed a model to help characterize the prophylactic benefit of the TPI preparation in mice.
The standardized endotoxic activity for two preparations of TPI was determined by the Associates of Cape Cod (Woods Hole, Mass.) using a Limulus amoebocyte lysate gel clot assay. Each preparation had equivalent endotoxin levels of 0.8 endotoxin unit/mg.
Four-week-old, female Swiss Webster mice were acclimated for 2 weeks as described previously (10). Mice comprising the control and treated populations were injected intraperitoneally with 0.1 ml of Salmonella enterica serovar Typhimurium ATCC 14028 (
5.00 x 103 bacteria per mouse) on day 0. Unless stated otherwise, treated mice were given a 5-mg subcutaneous injection of TPI at the time designated by the experimental protocol while control mice received a placebo of physiological saline. Negative control mice were sham handled in a manner similar to that of the control and treated populations to evaluate the influence of nonexperimental parameters on mortality. To obtain statistical significance, five mice were housed per cage and a minimum of five cages were used per treatment group. The mice were monitored daily, and mortality was recorded until 80% of the control mice died. All experiments were arranged in a completely randomized design and analyzed as described by Willeford et al. (10). A P value of less than 0.05 was necessary for the results to be considered significant.
The effect of varying the timing and dosage of TPI pretreatment on the survival of the mice in response to a potentially lethal challenge with Salmonella serovar Typhimurium was investigated. TPI was administered on day -4, -2, or -1 (days before challenge) or coincidently with the challenge on day 0 (Fig. 1A). Deaths were usually not observed until 4 days after the challenge in the control populations of female Swiss Webster mice challenged with Salmonella serovar Typhimurium. A rapid increase in death ensued, with approximately 80% mortality occurring 8 days postchallenge. Mice treated with TPI 4 days prior to challenge showed no significant difference in mortality from mice in the control group. A prophylactic benefit was observed, however, if TPI was given on either day -2 or day 0. By day 8, the mortality in the control population reached 80%, while groups that received TPI on day -2 and day 0 had mortality rates of 60 and 54%, respectively. Mice treated 1 day prior to challenge had the fewest deaths through 8 days postchallenge (32%). When the mortality rate was established in this control population, a statistically significant difference between mortality in the control group and the mortality in each of the groups treated with TPI on days -2, -1, and 0 became evident. The day -1 treatment group also had significantly lower mortality than the day 0 and day -2 TPI treatment groups, as exemplified by the day 8 P values of 0.0193 and 0.0014, respectively.
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FIG. 1. Control mice were given 0.1 ml (5 x 103 CFU) of Salmonella serovar Typhimurium intraperitoneally on day 0 ( ). Treated mice received both S. typhimurium on day 0 and either a 5-mg subcutaneous injection of TPI on day -4 ( ), day -2 ( ), day -1 (•), or day 0 ( ) (A) or an injection of TPI of 20.0 (), 15.0 (), 5.0 (•), or 0.1 () mg on day -1 (B). Each data point represents the average daily mortality (n = 5) associated standard exponential error (error bars) per cage of five mice.
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A Griess reagent kit (Molecular Probes, Eugene, Oreg.) was used to determine nitrite levels produced by stimulated and nonstimulated macrophages. RAW 264.7 cells (a murine alveolar macrophage line) were obtained from the American Tissue Culture Collection (Manassas, Va.; ATCC TIB-71) and cultured in a complete medium (Dulbecco's modified Eagle's medium) specified by the American Tissue Culture Collection. Cells were maintained at 37°C in a humidified incubator containing 5% CO2 and were plated at a density of 105 cells/cm2 in 24-well culture plates and allowed to adhere for 4 h. The medium was then removed and replaced with complete medium (control wells) or with complete medium supplemented with either TPI (0.1, 1, or 10 mg/ml) or a 50-µg/ml concentration of lipopolysaccharide (serotype O127:B8; Sigma, St. Louis, Mo.) for 72 h (n = 1). Separate plates were used for three time points (i.e., 24, 48, and 72 h). Supernatant (150 µl) was removed at the designated time and mixed with 20 µl of Griess reagent and 130 µl of double-distilled water in a 96-well tissue culture plate. The microplate was incubated at 37°C for 30 min, and the absorbance was read at 548 nm on a µQuant plate reader, with a standard curve generated with sodium nitrite.
Protein determinations of adherent macrophages were made to establish a standard for nitrite production. Supernatant was removed for nitrite analysis, and the wells were washed with sterile phosphate-buffered saline. Adherent cells were lysed for 30 min at room temperature by adding 1 ml of 0.3 M NaOH. A Bradford assay for microplates (Bio-Rad, Hercules, Calif.) was run in triplicate as described in the kit instructions, with bovine serum albumin in 0.3 M NaOH as the standard.
Macrophages stimulated by lipopolysaccharide produced a significant increase in nitrite production over that of the control cells by day 2 (671.1 versus 7.6 µmol of nitrite produced/mg of adherent protein, P < 0.0001). TPI did not stimulate macrophage activity when tested at concentrations up to 10 mg/ml (9.6 µmol of nitrite produced/mg of adherent protein on day 3). Neither cytotoxic nor proliferative effects due to the administration of TPI were observed.
Agents which retard pathogenesis may enable a host to mount a successful defense in response to challenges of the immune system. These agents can provide specific (i.e., in the form of antibodies) or general protection and can enhance overall immunocompetence. Cytokines and cationic peptides are two such classes of nonspecific defense agents. We isolated from caprine serum a mixture of three peptides which have an estimated molecular mass of approximately 5 kDa, as determined by size exclusion chromatography (data not shown), and which significantly reduced the ensuing mortality when administered to mice challenged with Salmonella serovar Typhimurium. The prophylactic benefit occurred in a dose-dependent manner, with a maximal effect garnered when approximately 15 mg of TPI (a total of 5.6 mg of protein) was administered. The benefit appeared to derive from TPI's proteinaceous components, in light of the observation that all benefit was lost after proteolytic digestion with bromelain and proteinase K (data not shown) or incubation at 85°C, procedures known to denature protein.
TPI showed no bactericidal activity when surveyed against a battery of both gram-positive and gram-negative bacteria (9) and did not contain a level of endotoxin sufficient to promote a pyrogenic response. However, its administration retarded bacterial pathogenesis in chickens (9) and mice, with the greatest survival rate observed when TPI was given approximately 24 h before challenge. A significant benefit was observed when TPI was given at the time of challenge or 2 days before challenge, but no benefit was observed when it was administered 4 days before a severe challenge.
Clonal and nonclonal immunity can be found in serum in the form of immunoglobins, collectins, cytokines, chemokines, and cationic peptides (8). Isolation of TPI effectively excludes globulins (
180 kDa), collectins (a collection of multimeric proteins with a subunit molecular mass of approximately 50 kDa), and cytokines (10 to 80 kDa) (5, 7). Chemokines (8 kDa) function mainly as chemoattractants for phagocytic cells, recruiting monocytes and neutrophils from the blood to sites of infection (8). Molecular mass estimates point more favorably towards a cationic peptide, some of which have masses as low as 2 kDa. Cationic peptides have been found to have diverse physiological roles in vivo (2, 4, 6) but are primarily associated with bactericidal activity with a MIC in the range of 1 to 100 µg/ml (1, 3). TPI at concentrations of over 2,500 times the upper MIC range recognized for cationic peptides produced no bactericidal activity against Salmonella serovar Typhimurium (9). Preliminary sequence data, however, show a significant presence of lysine and arginine, which is consistent with the nature of cationic peptides. The bioactive peptide(s) in TPI may initiate an atypical or unclassified response for a cationic peptide.
It is unclear whether administration of TPI affects the ability of the host to kill the bacteria, to control their subsequent rate of growth, or both. TPI, however, positively influences the ability of the host to withstand and survive the challenge of an infectious agent.
Studies are being pursued to elucidate TPI's mechanism of action. While TPI does not appear to work by directly stimulating macrophages, it remains possible that TPI causes indirect stimulation through the production of macrophage-activating effector molecules such as interferon or tumor necrosis factor.
This work was supported in part by Mississippi Agricultural and Forestry Experiment Station project no. 9807.
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Box 9650, Mississippi State University, Mississippi State, MS 39762. Phone: (662) 325-2651. Fax: (662) 325-8664. E-mail: KOWMSU{at}RA.MSSTATE.EDU.
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- Willeford, K. O., T. A. Parker, E. D. Peebles, C. Wang, and E. W. Jones. 2000. Reduction of mortality in specific-pathogen-free layer chickens by a caprine serum fraction after infection with Pasteurella multocida. Poult. Sci. 79:1424-1429.
[Abstract/Free Full Text] - Willeford, K. O., T. A. Parker, G. T. Pharr, and K. Buddington. 2001. Prophylactic effects of a caprine serum factor (CSF-1) in mice infected with Salmonella typhimurium. Drug Dev. Res. 54:45-51.
Antimicrobial Agents and Chemotherapy, June 2002, p. 1971-1973, Vol. 46, No. 6
0066-4804/02/$04.00+0 DOI: 10.1128/AAC;46.6.1971-1973.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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