Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity

Abstract

The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amoebocyte lysate (CLAL) assay. When bacterial cells exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring 0-hydroxymyristic acid (3-OH tetradecanoic acid, D-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli OlKl strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their I-OHC14:0 contents. Four other E. coli strains (0 serotypes 014, 024, and 075) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli OlKl and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the jI-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the 05 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.

The role of the Lps gene in experimental ulcerative colitis in mice

“…All DSS-treated groups showed significantly increased levels of circulating endotoxins ( Table 3), indicating that the colonic mucosal barrier had been damaged by the treatment. The circulating endotoxins may have originated from bacteria present in the intestinal lumen and in the inflamed intestinal tissue, as well as from those present in the livers and spleens (18). Most probably, only a minor fraction came from intact bacteria circulating in the blood, since bacteria are rapidly taken up by the mononuclear phagocytic system (31).…”

“…Endotoxins (lipopolysaccharide, LPS) in the cell wall of Gram-negative bacteria or as released cell wall fragments (18) are strong inflammatogenic components (21). LPS is one of the most potent stimulators of tumor necrosis factor alpha (TNFa), a major mediator of inflammation and lethality (21).…”

Inactivation of airborne bacteria using different UV sources: Performance modeling, energy utilization, and endotoxin degradation

“…Microbial inactivation through UVGI results from denaturation of enzymes, proteins, and membranes and disruption of cellular metabolic activities (Takashima et al, 2007). However, intracellular substances may be released when the cells in bioaerosols are damaged or destroyed (Mattsbybaltzer et al, 1991). Endotoxins are complexes of lipopolysaccharides (LPS), proteins, and hazardous biological substances ( Fig.…”

A non‐chromatographic method for the removal of endotoxins from bacteriophages

“…Ff filamentous (bacterio)phages (comprising M13, f1, and fd) are produced by infection of the (Gram-negative) bacterium E. coli and are showing promise as a new class of pharmaceutical therapeutics. The infection of the host is non-lethal; the phages are continuously extruded through the cell wall (Russel et al, 2004) but the resulting extracellular medium is still heavily contaminated with endotoxins by the end of growth (Jang et al, 2009;Mattsby-Baltzer et al, 1991). Filamentous phage-based products show potential in areas as diverse as gene therapy, vaccination, and biomaterials (Gonzalez et al, 2010;Solomon, 2008;Xu et al, 2012).…”