In 1977, Arnold and co-workers reported a bactericidal effect for native LF molecule, which apparently was distinct from the stasis mechanism. These experiments were performed with microbial cells suspended in deionized water or buffer solutions at acid pH and the reported mechanism is controversial. Various laboratories have failed to demonstrate a similar bactericidal effect (Rainard, 1987; Gutteberg et al., 1990). Lassiter (1990), from Arnold's group, later published a doctoral thesis which indicated that cont
Figure 5. Growth of S. typhimurium 395MS(S) (S; parent with smooth LPS) and its isogenic mutant Rd (with rough LPS) in SPYE broth. Control (A), with 1 mg/ml bLF (O) and 5 mg/ml bLF ( •). [Redrawn from Naidu, et al., 1993],
Figure 5. Growth of S. typhimurium 395MS(S) (S; parent with smooth LPS) and its isogenic mutant Rd (with rough LPS) in SPYE broth. Control (A), with 1 mg/ml bLF (O) and 5 mg/ml bLF ( •). [Redrawn from Naidu, et al., 1993], amination of EDTA during dialysis of LF could account for the cidal effect against E.coli. Furthermore, the cidal effects of LF against oral streptococci seem due to the acid pH of the test system. Degradation products in an LF preparation such as the cationic peptides could elicit membrane damage and kill microorganisms. In an antimicrobial milieu such as in the phagosome, LF could possibly elicit a cidal effect synergistic with oxidative events. However, clear evidence for a direct cidal effect with native (intact) LF molecule in vitro is still lacking. This section has reviewed the cidal effect in a chronological perspective with no endorsement for the mechanisms hypothesized in the literature.
LFs seem to elicit cidal effects against a variety of microorganisms including Gram-positive and Gram-negative bacteria, rods and cocci, facultative anaerobes, and aero-tolerant anaerobes. Similar morphological and physiological types are represented among the LF-resistant bacteria (Arnold et al., 1980). S. mutans was more resistant to LF when grown on a sucrose-containing medium than when it was grown on brain heart infusion broth without added sucrose. When an LF-sensitive, avirulent strain of Streptococcus pneumoniae was passed through mice, the resultant virulent culture demonstrated resistance to LF. Since organisms of the same species and even of the same strain such as S. pneumoniae, can differ in susceptibility to LF, it appears that accessibility to the LF target site may account for variations in susceptibility.
Influence of several physical conditions and the metabolic state of Streptococcus mutans on LF susceptibility were reported (Arnold et al., 1981). After exposure to LF, a 15-min lag period occurred before the initiation of killing, indicating that a two-step process is involved in LF killing. Cultures harvested during the early exponential phase were sensitive to LF, whereas cultures harvested in the early stationary phase were markedly resistant. The rate of killing was dependent on temperature; there was no loss of viability at 2°C. Killing occurred at pH 5.0 to 6.0 in water and 20 mM glycine, but not at any pH in 50 mM sodium phosphate or N-2 hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer. Addition of exogenous ferrous or ferric ions did not reverse or prevent LF killing, nor did 1 mM magnesium chloride.
Bactericidal effect of LF against Legionella pneumophila was reported (Bortner et al., 1986). Purified apo-hLF elicited cidal activity against L. pneumophila (serogroup 1), with a 4-log decrease in viability within 2 h at 37 °C. Guinea pig passage of this strain did not affect its sensitivity to LF. Addition of magnesium blocked the bactericidal activity. In addition, human milk was also cidal for L. pneumophila. Salts including CaCl2, Mg(N03)2, and MgCL, but not NaCl, blocked killing. Activity was pH dependent with the greatest activity at 5.0. Sensitivity of the organism was markedly affected by the growth conditions. Log-phase 12 h, broth-grown cells were most sensitive, with older cultures appearing more resistant. Plate-grown cells were completely resistant. LF binding, as detected by immunofluorescence microscopy, was temperature dependent (no binding was observed at 4°C), but was independent of killing (Bortner et al., 1989).
Actinobacillus actinomycetemcomitans is a fastidious, facultative Gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human LF is bactericidal for this pathogen (Kalmar & Arnold, 1988). This cidal activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log reduction in viability within 120 min at 37°C at a concentration of 1.9 |iM. Magnesium enhanced LF killing, while other cations, such as potassium and calcium, had no effect.
It was reported that selective anions were capable of inhibiting the expression of bactericidal activity by LF on S. mutans 10449 (Lassiter et al., 1987). The ability to block LF expression was directly related to the capacity of the anion to serve as a coordinate ion in iron-binding by the LF molecules. The authors hypothesized the presence of an anionic LF target site on the bacterial surface. Treatment of S. mutans with LF under anaerobic conditions abrogated the bactericidal effect. LF killing could be enhanced with thio-cyanate and inhibited by catalase and lactoperoxidase; however, bovine serum albumin was equally effective as an inhibitor.
Antimicrobial effects of LF and human milk on Yersinia pseudotuberculosis was reported (Salamah & al-Obaidi, 1995a). Bacterial growth in vitro was inhibited by apo-but not holo-LF or human milk. Iron-free human milk and to a lesser extent normal human milk were bactericidal for Y. pseudotuberculosis cells that were suspended in deionized water. The in vivo studies also showed that iron-saturated LF enhanced growth, whereas the viable count was reduced by iron-free LF and EDTA. Nine envelope proteins were decreased or disappeared upon growth in iron-deficient medium, whereas one new high molecular weight protein appeal ed under the same conditions. The effect of pH, temperature, concentration of magnesium and calcium on the bactericidal activity of LF against Yersinia pseudotuberculosis was investigated (Salamah & al-Obaidi, 1995b). The bactericidal activity of LF was higher at acid pH, whereas the bactericidal activity of TF was higher at alkaline pH. Both were not efficient at 4°, 15°, and 25°C, but were efficient at 37°C. LF, but not TF, was very efficient at 42°C. The activity of both proteins were time and concentration dependent. Calcium did not effect their activity up to 60 mM, whereas magnesium reduced the activity of LF only.
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