Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy

Abstract

Lipopolysaccharide (LPS), also referred to as endotoxin, is the major component of Gram-negative bacteria's outer cell wall. It is one of the main types of pathogen-associated molecular patterns (PAMPs) that are known to elicit severe immune reactions in the event of a pathogen trespassing the epithelial barrier and reaching the bloodstream. Associated symptoms include fever and septic shock, which in severe cases, might even lead to death. Thus, the detection of LPS in medical devices and injectable pharmaceuticals is of utmost importance. However, the term LPS does not describe one single molecule but a diverse class of molecules sharing one common feature: their characteristic chemical structure. Each bacterial species has its own pool of LPS molecules varying in their chemical composition and enabling the aggregation into different supramolecular structures upon release from the bacterial cell wall. As this heterogeneity has consequences for bioassays, we aim to examine the great variability of LPS molecules and their potential to form various supramolecular structures. Furthermore, we describe current LPS quantification methods and the LPS-dependent inflammatory pathway and show how LPS heterogeneity can affect them. With the intent of overcoming these challenges and moving towards a universal approach for targeting LPS, we review current studies concerning LPS-specific binders. Finally, we give perspectives for LPS research and the use of LPS-binding molecules.

Keywords: LPS-binding molecules; detection; endotoxin; immunology; lipid A; low endotoxin recovery.

Figure A1

Structures of lipid A from different bacterial species. References to structures are listed in Table 1. Possible variations of the structures are marked in red.

Figure A1

Structures of lipid A from different bacterial species. References to structures are listed in Table 1. Possible variations of the structures are marked in red.

Figure A1

Structures of lipid A from different bacterial species. References to structures are listed in Table 1. Possible variations of the structures are marked in red.

Figure A1

Structures of lipid A from different bacterial species. References to structures are listed in Table 1. Possible variations of the structures are marked in red.

Figure A1

Structures of lipid A from different bacterial species. References to structures are listed in Table 1. Possible variations of the structures are marked in red.

Figure A1

Structures of lipid A from different bacterial species. References to structures are listed in Table 1. Possible variations of the structures are marked in red.

Figure 1

Schematic illustration of the LPS structure. LPS shown in bright red is anchored in the outer membrane of (G-) bacteria. Smooth- (S), semi-rough- (SR), rough- (R) or deep-rough- (DR) LPS types are defined by the length of the O-antigen and core region. The variability decreases from the outermost part of LPS to the hydrophobic innermost part. Different sugar moieties are shown in blue and green; heptose is shown in yellow; 3-deoxy-D-manno-octulosonic acid (KDO) in orange, and glucosamine (I and II) in gray.

Figure 2

Overview of the main challenges in endotoxin quantification. The chemical variability of LPS, supramolecular structures, and interaction with molecules result in inaccurate and/or unreproducible endotoxin quantification measurements.

Figure 3

Scheme of the most relevant pathways for LPS detection in human cells. Left: detection of intracellular LPS via caspase proteins causes the assembly of a supramolecular complex called inflammasome, which induces the initiation of the potent inflammatory process called pyroptosis. Right: detection of extracellular LPS via the TLR4 receptor complex and initiation of inflammatory responses via the MyD88 and TRIF signaling cascades.

Figure 4

Schematic representation of Yersinia pestis life cycle with focus on how the lipid A moiety changes during the different stages, ultimately interfering in the host’s immune response. The top part of the image shows the life cycle in wild animals. Briefly, Yersinia pestis-infected fleas bite wild rodents and transmit the pathogen. Triggered by the rodent’s body temperature of 37 °C, it starts producing a less immunogenic variant of lipid A (tetra-acylated), which is not able to elicit an effective immune response. As a result, Yersinia pestis is not fully eliminated and persists. The rodent becomes a reservoir of the pathogen in the wild and can, in turn, infect vector fleas, which feed on their blood. Once Yersinia pestis reaches the flea digestive tract (27 °C), it produces a hexa-acylated form of lipid A and starts proliferation in these favored conditions. By biting a new host, the flea can further spread the disease. Once humans are accidentally bitten (bottom part of the figure), the bacterium produces the tetra-acylated form of lipid A again to adapt to the human body temperature. However, in contrast to rodent TLR4, the human TLR4 does not recognize this lipid A variant, and therefore, it is not able to activate the downstream signaling cascade. As a result, the immune response is ineffective as it has to rely only on other defense mechanisms. Yersinia pestis invades the body undisturbed, causing a disease called plague.

Figure 5

Graphical scheme depicting the underlying mechanisms in different LPS detection methods. From left to right: rabbit pyrogen test (RPT), monocyte activation test (MAT), gel clot limulus amebocyte lysate (LAL) assay and its variants (e.g., chromogenic LAL), EndoLISA, and, ultimately, the TLR4-NF-κB-luciferase (TLR4-NF-κB-luc) reporter gene assay.