Myeloperoxidase selectively binds and selectively kills microbes

Abstract

Myeloperoxidase (MPO) is reported to selectively bind to bacteria. The present study provides direct evidence of MPO binding selectivity and tests the relationship of selective binding to selective killing. The microbicidal effectiveness of H(2)O(2) and of OCl(-) was compared to that of MPO plus H(2)O(2). Synergistic microbicidal action was investigated by combining Streptococcus sanguinis, a H(2)O(2)-producing microbe showing low MPO binding, with high-MPO-binding Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa without exogenous H(2)O(2), with and without MPO, and with and without erythrocytes (red blood cells [RBCs]). Selectivity of MPO microbicidal action was conventionally measured as the MPO MIC and minimal bactericidal concentration (MBC) for 82 bacteria including E. coli, P. aeruginosa, S. aureus, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, and viridans streptococci. Both H(2)O(2) and OCl(-) destroyed RBCs at submicrobicidal concentrations. Nanomolar concentrations of MPO increased H(2)O(2) microbicidal action 1,000-fold. Streptococci plus MPO produced potent synergistic microbicidal action against all microbes tested, and RBCs caused only a small decrease in potency without erythrocyte damage. MPO directly killed H(2)O(2)-producing S. pyogenes but was ineffective against non-H(2)O(2)-producing E. faecalis. The MPO MICs and MBCs for E. coli, P. aeruginosa, and S. aureus were significantly lower than those for E. faecalis. The streptococcal studies showed much higher MIC/MBC results, but such testing required lysed horse blood-supplemented medium, thus preventing valid comparison of these results to those for the other microbes. E. faecalis MPO binding is reportedly weak compared to binding of E. coli, P. aeruginosa, and S. aureus but strong compared to binding of streptococci. Selective MPO binding results in selective killing.

FIG. 1.

Photographic demonstration of MPO binding and coloration of S. aureus, E. coli, and P. aeruginosa. Note the absence of MPO binding to S. sanguinis (viridans group). The four tubes to the left show the centrifuged microbe pellets in the absence of MPO exposure. The center tube contains 2 mg/ml MPO without microbes. The four tubes to the right show the centrifuged microbe pellets exposed to MPO.

FIG. 2.

Photograph of petri plates at the 10⁻³ CFU dilution used to measure S. sanguinis-MPO synergistic action against E. coli. The top and bottom rows of plates present the findings in the absence and presence of RBCs, respectively. From left to right, the final MPO concentrations were 0, 1.9, 5.6, and 50 nM. The plates were incubated for about 48 h to better visualize the smaller streptococcal colonies.

FIG. 3.

Photograph of petri plates at the 10⁻³ CFU dilution (top four and bottom left three plates) and 10⁻² CFU dilution (bottom far right plate) used to measure S. sanguinis-MPO synergistic action against S. aureus. The top and bottom rows of plates present the findings in the absence and presence of RBCs, respectively. From left to right, the final MPO concentrations were 0, 5.6, 16.7, and 50 nM. The plates were incubated for about 48 h to better visualize the smaller streptococcal colonies.

FIG. 4.

The composite results of MPO MIC and MBC testing with the data presented as Tukey box plots. The results are grouped by the medium used, i.e., CAMHB and CAMHB-5% LHB. Five groups of bacteria were tested. As indicated on the x axis, the groups (number of isolates) are as follows: Gram-negative E. coli (n = 6) and P. aeruginosa (n = 6), S. aureus (n = 16), E. faecalis (n = 7), Lancefield group A S. pyogenes (n = 10) and group B S. agalactiae (n = 10), and viridans group streptococci (n = 27). The bottom and top portions (hinges) of each box are the lower and upper quartiles, respectively, and the hinge or H-spread (i.e., the interquartile range) is the distance between the bottom and top of the box. The heavy horizontal band shows the median. The whiskers are the lines drawn from the upper hinge to the upper adjacent value and from the lower hinge to the lower adjacent value within 1.5× the H-spread. An outlier is marked with a circular dot if it is between the inner fences (i.e., 1.5× the H-spread) and outer fence (i.e., 3× the H-spread). An extreme outlier is marked by an asterisk if it is beyond the outer fences.