**3. Lipopolysaccharide structures and conformations**

discriminate pathogen from host flora. This requires an understanding of pathogen biology, the types of samples they occur in, and their mechanism of immune interaction within the hosts [1]. The innate immune system is able to discriminate pathogens from nonpathogens, and rap‐ idly sense pathogen biomarkers in the complex milieu of the host. Exploiting this recognition via measurement of pathogen signatures, can provide an optimal strategy for discriminatory biodetection. A primary category of such biomarkers is virulence signatures termed pathogen‐ associated molecular patterns (PAMPs) [2]. PAMPs are evolutionarily conserved molecules that bind pattern‐recognition receptors in the host, and activate the innate immune response [2, 3], providing a means for both early and specific pathogen detection. Biochemically, PAMPs are a diverse array of proteins, lipopeptides, lipoglycans, peptidoglycans, teichoic acids, and nucleic acids [4]. However, many detection methods have largely focused on proteins and nucleic acids [1, 5], ignoring other categories of PAMPs [2, 6–8]. Also, their small size, biochem‐ istry, and low concentration in hosts make them difficult to target in detection assays [8, 9].

142 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Classified as a lipogylcan, lipopolysaccharides (LPS) are small amphiphilic molecules that are associated with Gram‐negative bacteria [7, 10]. LPS is an indicator of active infection, is sero‐ group‐specific [11–13], more stable than its protein counterparts, and is released early in infec‐ tion, making it an ideal candidate for detection and diagnostics. LPS serves as a biomarker that aids in serological discrimination of Gram‐negative bacteria; this allows for identification and characterization of pathotypes that are essential for timely mitigation and treatment of infec‐ tions. Since LPS is a pathogen‐specific biomarker, it is an indicator of acute infection, which is an advantage over serological assays. In addition to medical diagnostics, LPS detection pro‐ vides a method for detecting *Escherichia coli* in the food‐industry, which is often associated with food‐borne illnesses. Finally, LPS is also a virulence factor whose structure and function deter‐ mines *E*. *coli* serogroup, a factor which has ramifications on vaccine design and therapeutic interventions. While many methods for LPS detection exist, most of them are not optimized for amphiphilic detection in physiological samples. An ideal measurement for LPS should be sen‐ sitive enough to detect low concentrations of the amphiphile in aqueous physiological milieu (e.g., blood), and use antibodies or ligands that provide serogroup selectivity [14]. Coupling sensitive detection platforms with surfaces designed to maximize the binding of amphiphilic

Bacteria are classified into Gram‐negative and Gram‐positive [15], which release amphiphi‐ lic virulence factors such as LPS, lipoarabinomannan (LAM), and lipoteichoic acid (LTA) in the host. Species of pathogenic Gram‐negative bacteria of concern to human health, include *Acinetobacter* [16], *Burkholderia* [17], *Bordetella* [18], *Campylobacter* [19–21], *Chlamydia* [22, 23], *E*. *coli* [20, 24], *Helicobacter* [25, 26], *Hemophilius* [27], *Klebsiella* [28], *Legionella* [20, 29], *Moraxella* [30], *Neisseria* [31], *Pseudomonas* [32], *Proteus* [33], *Salmonella* [20, 34], *Shigella* [35], *Yersinia* [36], and others, grouped into the Enterobacteriaceae family. These pathogens are contaminants in food, water, and soil, used as agents of bioterrorism, and can cause nosocomial infections [5]. Detection of these organisms, particularly *E*. *coli*, is an important aspect for epidemiology,

PAMPs is a potential solution to achieve such an ideal.

**2. Sources of lipopolysaccharides**

disease control, and treatment.

Lipopolysaccharides have been the subject of intense study for over half a century [37–39]. LPS is the prototypical lipoglycan with an overall net negative charge [40–42], and is the primary component of the outer membrane of nearly all Gram‐negative bacteria [11]. The bacterial membrane of each *E*. *coli* cell is composed of approximately 106 lipid A moieties and 107 glycerophospholipid molecules, comprising approximately three‐quarters of the outer membrane [43–45]. Thus, there are approximately 62 pg of LPS per cell (for *E*. *coli* in log phase growth) [46]. LPS has an amphipathic tripartite structure (**Figure 1**). Lipid A is the most conserved portion of the LPS molecule, and consists of six, sometimes seven, fatty acid tails (*E*. *coli* and *Salmonella*, respectively), which gives the molecule its hydrophobic properties [10, 43, 45]. Lipid A is also called endotoxin [43], and is responsible for the biological effects of LPS

**Figure 1.** Representative structure of the molecular components of smooth LPS. The hypervariable O‐polysaccharide antigen, core polysaccharide, and the hydrophobic lipid A group. Reprinted with permission from Ref. [74].

caused by its binding to the mammalian innate immune receptor, toll‐like receptor 4 (TLR4) [11, 44, 47, 48]. Structurally, lipid A is covalently bound to the core polysaccharide, which is further divided into the inner and outer core polysaccharides, with the outer core being less conserved in both sugar moieties and location of glycosidic linkages [45, 49, 50].

micelle assembly [10, 59, 79, 80]. Other factors that contribute to micelle shape [10, 79] are pH [61], ion concentration [81–86], and temperature [62]. These biochemical properties drive host‐

Detection Methods for Lipopolysaccharides: Past and Present

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pathogen interactions and should be considered in the design of detection strategies.

of sensitivity, but many lack the ability to differentiate between LPS serogroups.

**4.1. Limulus amoebocyte lysate assay and the rabbit pyrogen test**

some parenteral devices [10].

the rabbit pyrogen test [46].

tive sensitivities of the LAL assay is shown in **Table 1**.

**4. Detection methods for lipopolysaccharides and similar amphiphiles**

There have been many efforts to establish rapid and reliable detection methods for LPS in clinical samples [10, 46] and for testing pharmacological products such as infusion fluids, sterile injectables, medical device implants, and others [87]. These methods can be broadly divided into six overlapping categories: *in vivo* and *in vitro* tests, immunoassays and their derivatives; biological, chemical, and cell‐based sensors. These methods span a broad range

The first method approved by the US Food and Drug Administration for LPS detection was called the rabbit pyrogen test [88–90], which simply measures the ability of an endotoxin to induce fever in an animal. Any febrile response was attributed to the presence of endotoxin [89–91]. The test, clearly, is activity‐based, and nonspecific. In the case of Hepatitis B vaccine manufacturing, the rabbit pyrogen test is still the standard method for determining endotoxin contamination [91], but the test is cost prohibitive and is minimally utilized today, except in

In 1956, Bang discovered that amoebocytes from *Limulus polyphemus* (a.k.a. horseshoe crab) agglutinate upon addition of endotoxin [46], as a result of a protease cascade [10]. Bang and Levin [46, 92] subsequently used this concept to devise a method for endotoxin detection. Since the lysates of amoebocytes were required, it was called the limulus amoebocyte lysate (LAL) assay, and is the gold standard for the detection of lipid A. The LAL assay is prone to variability and can be inhibited through several mechanisms. The United States Pharmacopeia and the Code of Federal Regulations have consequently published guidances for the manu‐ facturing and testing of assays for use on human products [93, 94]. Despite some challenges, the LAL assay is more rapid, cost effective, and reportedly 300 times more sensitive [46] than

Variants of the LAL assay use turbidimetric [95], chromogenic [46], or viscosity [10] measure‐ ments to determine results [10, 46]. A turbidimetric gel clot has more coagulen, and measures the change in turbidity over time, but does not form a solid clot [46, 95]. The viscosity assay, however, measures the degree of clotting via the change in viscosity. The chromogenic assay can be endpoint or kinetic, and utilizes a *p*‐nitroaniline substrate, which is cleaved by an LAL proenzyme, providing a colorimetric readout [46]. The sensitivity of LAL assays is depen‐ dent on the sample type, processing method and time, as well as the dilution factor [46]. Additionally, the source of the LAL reagent plays a factor, as it is apparent when comparing the different limits of detection (LoD) reported for endotoxin standards. A survey of the rela‐

There are two main forms of LPS—smooth (S‐form) and rough (R‐form) [42, 45, 46]. The distal end of LPS extends to a long chain O‐polysaccharide antigen (O‐ag(s)) in organisms possess‐ ing S‐form, which is an indicator of virulence [51, 52]. R‐form LPS is devoid of the O‐ag [45], but can still induce an immunogenic response [53]. The O‐ag is hyper‐variable, and made up of repeating subunits, each composed of 1–7 glycosyl residues [45, 54]. As many as 40 size variations in subunit repeats of the O‐ag have been reported just for *E*. *coli* O111:B4 [55], and 180 O‐ag have been identified overall for *E*. *coli* species [47, 54]. The sugars (colitose, paratose, tyvelose, and abequose) that make the O‐ag unique are seldom found elsewhere [54]. Other variations to the polysaccharide are implemented through addition of noncarbohydrate enti‐ ties, such as acetyl or methyl groups [54]. These variations make discriminative detection of enteric bacteria feasible [56], but complicate antigen characterization. Therefore, LPS serves as an ideal target for early detection and identification of Gram‐negative pathogens.

In aqueous solutions, amphiphiles like LPS can present in a micellar conformation [48, 55, 57–59]. This occurs at a concentration specific to the amphiphile [55], and is known as the criti‐ cal micelle concentration (CMC). At or above the CMC, there is an equilibrium state between monomers, micelles or supramolecular aggregates, depending on environmental conditions [48, 55–57, 60–63]. This amphiphilic biochemistry and structural variability complicates determination of the exact molecular weight of S‐form LPS. As such, LPS concentrations are reported in weight per volume, or in endotoxin units (EU), a measure of activity. As degree of endotoxicity can vary according to bacterial origin, a rough estimate of 100 pg = 1 EU is used in many cases to facilitate unit conversion [64, 65].

The large oligosaccharide region on S‐form LPS makes the molecule amphipathic [54], which influences the shape of micelles in solution. Lipid A is largely responsible for shaping the LPS micelle [10, 45, 46, 56, 66–68], although other factors can also contribute. Lipid A is con‐ served within species in the number of fatty acid chains and the degree of saturation [44, 66] within those chains [22, 47, 69]. Shapes for LPS micelles include cubic, lamellar, and hexago‐ nal inverted structures [56, 67, 70, 71]. Whether aggregate or monomeric forms (or both) of LPS is required for innate immune activation is debatable [56, 72, 73]. Since this process occurs in aqueous blood, it is unlikely that the molecule is presented as a monomer, unless associated with serum binding proteins.

Variation in LPS micelles [55] modifies presentation of O‐ag‐specific epitopes to antibodies, making detection challenging [74, 75]. This is specifically true when the heterogeneous pre‐ sentation of linear [76] and conformational epitopes [49, 77] present on LPS molecules are con‐ sidered. The primary structure of LPS varies in the core polysaccharide, within and between species [47, 55]. Core polysaccharides are primarily made up of common sugars such as heptose and 2‐deoxy‐d‐*manno*octulosonic acid (a.k.a. KDO), which can be functionalized with phos‐ phate or ethanolamine groups [45, 50, 78]. This feature contributes to varying charge distribu‐ tions and differential size ratio of the hydrophobic to hydrophilic regions which influences micelle assembly [10, 59, 79, 80]. Other factors that contribute to micelle shape [10, 79] are pH [61], ion concentration [81–86], and temperature [62]. These biochemical properties drive host‐ pathogen interactions and should be considered in the design of detection strategies.
