[The neutralizing effect of histidine-rich-polypeptides on LPS from suspected periodontal pathogens]

Human parotid histidine-rich-polypeptides (HRPs) are a family of lowmolecular-weight, cationic polypeptides. HRP-1, HRP-3 and HRP-5 comprise 85%-90% of the total HRPs and are called major HRPs. There were many researches indicating the antimicrobial activities of HRPs. Recently, HRPs were reported to have an inhibitory action on the lipopolysaccharide (LPS) of E. coli, with HRP-5 being the most effective one among the three major HRPs. Since the LPS of oral gram-negative bacteria is thought to be one of the important etiological factors during the development of periodontal diseases, our experiment was aimed to investigate the neutralizing effect of human parotid HRP-5 on the LPS of anaerobic suspected periodontal pathogens, which have different chemical structures and biological activities compared with LPS of aerobic E. coli.

By using of preparative acid urea polyacrylamide gel electrophoresis (AU-PAGE), HRP-5 was purified from parotid saliva collected from healthy adults. Two stains of suspected periodontal pathogens, Porphyromonas gingivalis (P.g, 47-A) and Fusobacterium nuceatum (F. n, separated from subgingival plaque of a patient with adult periodontitis) were mass cultured. After harvested in the midlogarithmic phase, they were washed and lyophilized. The LPS of dried bacteria were extracted by the modified Westphal hot phenol-water procedures and purified by the enzyme digestion plus ultracentrifugation. Limulus test was applied to test the neutralizing effect of HRP-5 on the LPS-induced gelation of Limulus amoebocyte lysate. In brief, the standard LPS of E. coli, or extracted LPS of P. g or F. n, was preincubated with HRP-5 in a tube separately in room temperature for 10 minutes. Then the reagent of Limulus amoebocyte lysate was added in all the tubes, continued the incubation in 37 degrees C for one hour. After that, the gelation level of every tube was observed.

LPS extracted from P. g and F. n both showed good purity and strong activities to induce gelation of Limulus amoebocyte lysate. The gelation induced by LPS (1 ng/ml) of these two anaerobic suspected periodontal pathogens were weakly inhibited by HRP-5 (10 micrograms/ml), similar to that observed with standard LPS of E. coli. To get a complete neutralizing effect on LPS, it may be important to increase the concentration of HRP-5.

It appeared that HRPs could neutralize the endotoxic properties of LPS of suspected periodontal pathogens, therefore may contribute to periodontal health. The present investigation further confirmed that HRPs are important components of the host non-immune defense system.