Antimicrobial peptides are an abundant and diverse group of molecules that are produced by many tissues and cell types in a variety of invertebrate, plant and animal species. Their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of antimicrobial peptide activity, their relevance to how peptides damage and kill microorganisms still need to be clarified. Recently, there has been speculation that transmembrane pore formation is not the only mechanism of microbial killing. In fact several observations suggest that translocated peptides can alter cytoplasmic membrane septum formation, inhibit cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis or inhibit enzymatic activity. In this review the different models of antimicrobial-peptide-induced pore formation and cell killing are presented.

Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?

Molecular basis of bacterial outer membrane permeability.

The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.

Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.

There are four major categories of diarrheagenic Escherichia coli: enterotoxigenic (a major cause of travelers' diarrhea and infant diarrhea in less-developed countries), enteroinvasive (a cause of dysentery), enteropathogenic (an important cause of infant diarrhea), and enterohemorrhagic (a cause of hemorrhagic colitis and hemolytic uremic syndrome). Besides manifesting distinct clinical patterns, these categories of E. coli differ in their epidemiology and pathogenesis and in their 0:H serotypes. Common features (albeit distinct for each category) include plasmid-encoded virulence properties, characteristic interactions with intestinal mucosa, and elaboration of various types of enterotoxins or cytotoxins. A less-well-defined fifth category of diarrheagenic E. coli is that of enteroadherent E. coli, so far identifiable only by their pattern of adherence to Hep-2 cells in tissue culture.

Escherichia coli that Cause Diarrhea: Enterotoxigenic, Enteropathogenic, Enteroinvasive, Enterohemorrhagic, and Enteroadherent

Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as “endotoxin protein,” suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ ( Lps d ) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.

Cutting Edge: Repurification of Lipopolysaccharide Eliminates Signaling Through Both Human and Murine Toll-Like Receptor 2

Es werden zwei einfache Verfahren zur Extraktion von Bakterien mit Phenol/Wasser angegeben. Nach Behandlung von gramnegativen Bakterien mit Phenol/Wasser-Emulsionen in der Kälte während weniger Minuten erhält man in der wäßrigen Phase die somatischen Glykoproteide der Bakterien (hauptsächlich 0-Antigene) in praktisch quantitativer Ausbeute. Nach Extraktion der Bakterien mit erwärmten, homogenen Phenol/Wasser-Mischungen und Trennen der Phasen in der Kälte finden sich in der wäßrigen Phase die proteinfreien Polysaccharide neben Nucleinsäuren. Einige chemische und immunologische Eigenschaften der nach den beiden Verfahren dargestellten Glykoproteide und Polysaccharide werden beschrieben.

Über die Extraktion von Bakterien mit Phenol/Wasser

Publisher Summary Lipopolysaccharides (LPS) form a large, unique class of macromolecules representing a characteristic attribute of gram-negative bacteria. Associated with proteins, they are located in the outer leaflet of the outer membrane of the bacterial cell. In this exposed position on the cell surface, lipopolysaccharides are involved in the interaction of the cell with the environment. Thus, contact of the bacterium with the immune system leads to the stimulation of specific antibodies directed predominantly against determinant structures of the lipopolysaccharide. Hence, lipopolysaccharides represent the main surface antigens of gram-negative bacteria. The chapter describes general aspects of the O-specific chains and the core, and principles of their biosynthesis. The O-specific chains of lipopolysaccharides are made up of repeating units of identical oligosaccharides. These units usually contain different constituents, thus the O chain represents a heteropolysaccharide. The chapter discusses the structure of Salmonella lipopolysaccharides (lipid A), and its biosynthesis. Lipid A's of other gram-negative bacteria, recently investigated is also described. The smallest lipid A substructure exhibits antigenicity, mitogenicity, lethal toxicity, (weak) pyrogenicity, and (weak) complement reactivity, but strong Limulus lysate activity. Some aspects of the biological properties of lipopolysaccharides are also discussed. In Gram-positive and Gram-negative bacteria, cell wall components other than lipopolysaccharide may also be endowed with endotoxin-like activities. Like lipopolysaccharide, these constituents are amphipathic in nature (probably with the exception of murein, though it may contain lipoprotein). Thus, the studies indicate that at least some lipid A activities are not restricted to one specific structure, but are rather connected with general physicochemical properties.

Lipopolysaccharides of Gram-Negative Bacteria

CHAPTER 4 – Isolation and Chemical and Immunological Characterization of Bacterial Lipopolysaccharides

A study on the presence of Escherichia coli 0111, 055, and 026 hemagglutinins and agglutinins in the sera of children and adults selected at random yielded the following results. The E. coli hemagglutination test proved to be far more sensitive for the detection of these antibodies than the conventional bacterial agglutination test. A relationship was found to exist between the presence of E. coli hemagglutinins and the age of the individuals; these antibodies were found in over 90 per cent of human sera selected at random from individuals 12 years of age and older, and less than 14 per cent of sera from infants up to the age of 3 months. Placental transfer of E. coli hemagglutinins was demonstrated in only 3 out of 26 cases, and in these instances the maternal antibody titers were from 4 to 16 times higher than those of the cord blood. These E. coli hemagglutinins were demonstrated also with red blood cells modified with purified lipopolysaccharides, indicating that they react with the somatic antigens of these serogroups of E. coli. The specificity of the hemagglutinins was established by absorption tests, utilizing red blood cells modified by one or the other of the lipopolysaccharides. E. coli 0111, 055, and 026 hemagglutinins were demonstrated in 2 commercial gamma globulin preparations, indicating that these antibodies are not restricted to a population in a small geographical area. The potential diagnostic usefulness of the E. coli hemagglutination test deserves further investigation.

Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages.

Aus Colibakterien (Stamm Kröger 0- Gruppe 8) wurde ein hochwirksames Pyrogen dargestellt. Die Bakterien wurden mit Phenol/Wasser extrahiert (Verfahren nach Westphal, Lüderitz u. Bister), die erhaltene wäßrige Lösung der Polysaccharid/ Nucleinsäure-Fraktion wurde zur Entfernung der Nucleinsäure mehrfach mit Äthanol fraktioniert; aus der so gewonnenen angereicherten Polysaccharid-Fraktion konnte durch Ultrazentrifugation das reine Pyrogen in einer Ausbeute von 1-2% der bakteriellen Trockensubstanz als Sediment abgetrennt werden. Es handelt sich um ein hochmolekulares, wasserlösliches Lipopolysaccharid, welches zu ~ 75% aus einer phosphorylierten (6-7% organisch gebundenes Phosphat) Polysaccharid-Komponente und zu 12-13% (Minimalwert) aus einer lipoidalen Komponente aufgebaut ist. Das Material ist frei von Protein-, Peptid- oder Aminosäure-Anteilen sowie von Nucleinsäure. Die pyrogene Grenzdosis nach intravenöser Injektion beim Kaninchen beträgt 0,002 μg, kg, beim Menschen 0,001 μg/kg. Es handelt sich um das bislang wirksamste Pyrogen, welches aus gramnegativen Bakterien isoliert werden konnte.

Otto Lüderitz

Papers

Über die Extraktion von Bakterien mit Phenol/Wasser

Lipopolysaccharides of Gram-Negative Bacteria

CHAPTER 4 – Isolation and Chemical and Immunological Characterization of Bacterial Lipopolysaccharides

Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages.

Über bakterielle Reizstoffe I. Mitt.: Reindarstellung eines Polysaccharid-Pyrogens aus Bacterium coli