α-Defensins are found in various leukocytes and in the Paneth cells of the small intestines of many mammals. Although human neutrophils contain four α-defensins, HNP1 to HNP3 are collectively about 60 times more abundant than HNP4 (6
). Largely because it has been difficult to obtain HNP4 from natural sources, this defensin has been studied less than HNP1 to HNP3. Wilde et al. (21
) used size-exclusion and reverse-phase high-performance liquid chromatography to purify native HNP4 from human neutrophils and then tested its antimicrobial activity. They asserted, “compared to a mixture of the other human defensins, HNP4 was found to be approximately 100 times more potent against E. coli
and four times more potent against both Streptococcus faecalis
and Candida albicans
). Our study provides partial support to their findings with E. coli
. The relative potencies of two peptides, e.g., HNP4 and HNP1, can be estimated from the ratio of their vLDs. When we used the mean vLD90
s shown in Table for E. coli
ATCC 8739, these ratios were 3 for HNP1, 4 for HNP2, and 13 for HNP3, signifying that HNP4 is approximately 3- to 13-fold more potent on a weight basis. If we used the mean vLD90
ratios for the other E. coli
strain, ATCC 25922, the ratios were 2 for HNP1 and HNP2 and 5 for HNP3. Had we chosen to use the vLD99
s for E. coli
ATCC 8739, they would have returned ratios that were two- to fourfold higher than the vLD90
ratios: 15 for HNP1 and 23 for HNP2 and HNP3.
Human neutrophils may have compensated for the lower intrinsic potencies of HNP1 to HNP3 against gram-negative bacteria by producing these peptides at a 60-fold greater abundance. The greater intrinsic potency of HNP4 against gram-negative bacteria evidently came with a price, since it was less potent than HNP1 and HNP2 against the gram-positive test organisms in our panel. In contrast, HD5 was as active as (or more so than) HNP4 against gram-negative bacteria, and it was almost as effective as HNP2 against the gram-positive organisms.
Perhaps the greatest surprise from our study was the exceedingly poor performance of HD6 against our bacterial test panel. Indeed, the relative inactivity of HD6 against bacteria initially led us to question its raison d'être. However, subsequent data from our group indicated that HD6 can inhibit infection of peripheral blood mononuclear cells by human immunodeficiency virus type 1 as effectively as HNP1 to HNP4 (unpublished data), suggesting that HD6 may have evolved specificity for viruses at the expense of its antibacterial activity. In the future, HD6 (and other intestinal defensins) deserve to be tested for their activities against anaerobes, fungi, protozoans, and helminths and for other properties within the purview of defensins, e.g., chemotaxis, adjuvanticity, and enhancement of wound healing (11
The conspicuous diversity of the survival curves shown in Fig. was also unexpected. Most defensins tested against gram-positive strains, including all six defensins against B. cereus
, consistently produced concave-down dose-response relationships that follow simple exponential killing (14
). Each curve that appears entirely concave down, as plotted on the log-log scale of Fig. , is linear on a lognormal plot (data not shown). This type of result would suggest that neither saturation by increasing defensin concentrations nor resistance to defensins influences the activity measured. By contrast, HNP4 produced bimodal curves against both S. aureus
strains, consistent with a small (<1:100) subpopulation of organisms that were phenotypically resistant to defensins rather than genotypically resistant (1
), especially since we used cloned organisms in each experiment. Similarly, bimodal curves were observed with HNP4 and HD5 against gram-negative strains E. coli
ATCC 8739 and E. aerogenes
. With the exception of the scant activity of HD6, none of the defensins tested against gram-negative strains produced strictly exponential survival curves. When deviations from a linear lognormal survival curve exist, only the most linear portions of the curve should be used to calculate relative potency. The human α-defensins are considerably less potent than antimicrobial peptides, such as ovine SMAP-29, porcine protegrins, or the horseshoe crab's tachyplesins. Although the vLD90
s and vLD99
s of human defensins are reliable, vLD99.9
s could be calculated for only one organism in our test panel. When more potent antimicrobial peptides (e.g., SMAP-29, protegrins, and tachyplesins) are assayed, reliable vLD99.9
s can also be obtained.
With these caveats, the virtual colony counting assay described in this report can produce results comparable to those of the NCCLS-approved colony counting procedures with far less effort. Our virtual constants were so named to distinguish them from any existing published constants while making their meaning readily understandable to workers in various fields of study. We do not intend to imply that our vLDs have been rigorously verified to correlate with LDs determined by other methods. However, our findings do appear to correlate well with those produced by colony counting. As expected, the mean vLD50
s reported in Table were slightly greater than the LD50
s reported previously (22
) in a colony counting study of the same preparations of these six defensins against E. coli
ATCC 25922 and S. aureus
ATCC 29213 with 3-h defensin exposure times. Colony counting data comparable to the remainder of the data in Table have yet to be reported, but our results agree generally with what is known about these peptides. For instance, in our study HNP3 was less active than HNP1 and HNP2 against all six strains, which agrees with the findings from studies that used a variety of methods (4
). Furthermore, the calibration data shown in Table and Fig. satisfied each of the assumptions underlying the use of growth kinetic curves to calculate C
, as specified by Brewster (1a
), i.e., the curves were parallel and evenly spaced, with similar slopes for all thresholds and excellent linear regression r2
values. Any error added by using this calibration procedure to enumerate small concentrations of bacteria is insignificant. The growth rates in the experimental wells with the same strain were similar, but they were not as close to identical as those for the strain in the calibration growth curves (Fig. ) or the control wells. Threshold time variations were minimized by averaging the results of experiments performed on 3 days and by choosing a low threshold ΔOD650
Several technical details were crucial in the development of the virtual colony counting assay. A potential drawback of using 96-well plates for a 14-h experiment is that moisture can condense on the inner surface of the lid. Brewster (1a
) applied detergent to the insides of 96-well lids before their use to deter the accumulation of hanging droplets. We found this step to be unnecessary, perhaps because we did the experiments in a 37°C room or used a different instrument for the measurements. Evaporation of liquid from the edge wells caused us to limit the collection of experimental data to the internal 60 wells, none of which decreased in volume by more than 3% when the volumes were measured after overnight experiments. Antimicrobial activity must be antagonized in order to allow cells to grow exponentially after the addition of twice-concentrated media. Inhibition of defensin activity was likely due to the carbohydrates present in the growth medium. MH II powder is composed of Casamino Acids; 14% (wt/wt) beef extract, which contains a variety of carbohydrates; and 7% (wt/wt) starch. Defensins are known to bind to various polysaccharides, including protein glycosyl moieties (20
), cellulose, and dextran sulfate, with high affinities. Virtual colony counting could be adapted to measure the activities of agents other than defensins if their antimicrobial activities are also strongly inhibited by 2× MHB itself or if a peptide binder, such as dextran sulfate, that does not slow bacterial growth is added to the MHB. Virtual colony counting is also adaptable to address hypotheses involving high salt concentrations, various divalent cation concentrations, or physiological conditions by supplementing the low-salt 10 mM sodium phosphate incubation buffer as desired.
Our virtual colony counting procedure allows the convenient, concurrent comparison of the antibacterial specificities of the human α-defensins. In addition, libraries of mutated defensins could be screened at one (or more) critical concentration(s) to deduce sequence-to-activity relationships, test mechanistic hypotheses, or identify candidate therapies that can be used to combat pathogenic bacteria. The assay may also have potential as a general method for determination of cationic peptide antibacterial activity.