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Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jacobsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Enzyme-linked immunosorbent assays (ELISAs) are widely employed for the detection of protein targets due to their ease of use, sensitivity, and potential for high-throughput analyses. However, the use of ELISAs to detect non-protein targets such as lipids and amphiphiles is complicated by the physical properties of these molecules, which affects their association with functional surfaces and recognition ligands. Here, we developed a unique lipoprotein capture ELISA in which the natural association between lipoproteins and amphiphilic molecules facilitates detection of the target biomarker in a physiologically relevant conformation. An assay to detect the glycolipid lipoarabinomannan (LAM), a cell membrane component and virulence factor associated with Mycobacterial infections, was developed as a proof of concept., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion. The lipoprotein capture assay relies on capture of the host lipoproteins, exploiting the biological association of host lipoprotein with microbial amphiphilic biomarkers to "concentrate" LAM. In contrast, the membrane insertion assay is independent of the association between pathogen amphiphiles and host lipoprotein association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane, which forms the functional surface of an optical biosensor. In this manuscript, we explored the use of these assays for the detection of LAM in sera from adults whose tuberculosis status had been well-characterized using conventional microbiological tests, and endemic controls. Using the lipoprotein capture assay, LAM signal/noise ratios were >1.0 in 29/35 (83%) individuals with culture-confirmed active tuberculosis, 8/13 (62%) individuals with tuberculosis symptoms, but no positive culture for M. tuberculosis, and 0/6 (0%) symptom-free endemic controls. To evaluate serum LAM levels without bias associated with potential differences in circulating host lipoprotein concentrations between individuals, we subsequently processed available samples to liberate LAM from associated host lipoprotein assemblies followed by direct detection of the pathogen biomarker using the membrane insertion approach. Using the membrane insertion assay, signal/noise for detection of serum LAM was greater than that observed using the lipoprotein capture method for culture-confirmed TB patients (6/6), yet remained negative for controls (2/2). Taken together, these results suggest that detection of serum LAM is a promising TB diagnostic approach, but that further work is required to optimize assay performance and to decipher the implications of LAM/host lipoprotein associations for diagnostic assay performance and TB pathogenesis., Competing Interests: Scientists from the Los Alamos National Laboratories, operated by the Triad LLC, that are authors on this manuscript, do not have competing interests, and are not consultants for any competing interests. Ramamurthy Sakamuri was a post-doctoral researcher at Los Alamos National Laboratory when he conducted this research. He has subsequently transitioned to Bako Diagnostics, which is his current affiliation. However, Bako Diagnostics did not participate or were not involved in the research presented in this manuscript in any way. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Bacteremia is a leading cause of death in sub-Saharan Africa where childhood mortality rates are the highest in the world. The early diagnosis of bacteremia and initiation of treatment saves lives, especially in high-disease burden areas. However, diagnosing bacteremia is challenging for clinicians, especially in children presenting with co-infections such as malaria and HIV. There is an urgent need for a rapid method for detecting bacteremia in pediatric patients with co-morbidities to inform treatment. In this manuscript, we have developed and clinically validated a novel method for the direct detection of amphiphilic pathogen biomarkers indicative of bacteremia, directly in aqueous blood, by mimicking innate immune recognition. Specifically, we have exploited the interaction of amphiphilic pathogen biomarkers such as lipopolysaccharides (LPS) from Gram-negative bacteria and lipoteichoic acids (LTA) from Gram-positive bacteria with host lipoprotein carriers in blood, in order to develop two tailored assays - lipoprotein capture and membrane insertion - for their direct detection. Our assays demonstrate a sensitivity of detection of 4 ng/mL for LPS and 2 ng/mL for LTA using a waveguide-based optical biosensor platform that was developed at LANL. In this manuscript, we also demonstrate the application of these methods for the detection of LPS in serum from pediatric patients with invasive Salmonella Typhimurium bacteremia (n = 7) and those with Staphylococcal bacteremia (n = 7) with 100% correlation with confirmatory culture. Taken together, these results demonstrate the significance of biochemistry in both our understanding of host-pathogen biology, and development of assay methodology, as well as demonstrate a potential new approach for the rapid, sensitive and accurate diagnosis of bacteremia at the point of need.

Recognition of Pathogen-associated Molecular Patterns (PAMPs) by Toll-like receptors is central to innate immunity. Many bacterial PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid have amphiphilic properties. The hydrophobicity of amphiphilic PAMPs contributes to increasing entropy and causes these molecules to self-aggregate or bind host carrier proteins in aqueous physiological environments. The goal of this work was to determine how innate immune signaling is impacted by physical presentation and association of amphiphilic PAMPs with serum carrier proteins, using LPS as an example molecule. Specifically, we measured LPS-induced cytokine profiles in murine macrophages when the antigen was presented associated with the various serum carrier proteins in serum versus a serum-depleted system. Our study demonstrates that the observed cytokine profiles are dramatically different when LPS is presented in buffer, versus in serum when it is associated with proteins, specifically with respect to inhibition of pro-inflammatory cytokines in the latter. These studies suggest that LPS-mediated cytokine expression is dependent on its presentation in physiological systems. The amphiphilicity of bacterial PAMPs and consequent association with lipoproteins is a feature, which should be taken into account in the design of in vitro experiments. Further studies of the interdependencies of different serum carriers can identify pathways for drug delivery and diagnostics., Competing Interests: The authors have declared that no competing interests exist.

Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM. We also profile the change in the expression of these TB biomarkers as a function of time from a small set of samples collected from studies of bovine TB-infected cattle. We demonstrate for the first time the direct detection of bovine LM in serum, and clearly show that the biomarker is expressed in detectable concentrations during the entire course of the infection.

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.

Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC., (Published by Elsevier B.V.)

Supported lipid bilayers have proven effective as model membranes for investigating biophysical processes and in development of sensor and array technologies. The ability to modify lipid bilayers after their formation and in situ could greatly advance membrane technologies, but is difficult via current state-of-the-art technologies. Here we demonstrate a novel method that allows the controlled post-formation processing and modification of complex supported lipid bilayer arrangements, under aqueous conditions. We exploit the destabilization effect of lipopolysaccharide, an amphiphilic biomolecule, interacting with lipid bilayers to generate voids that can be backfilled to introduce desired membrane components. We further demonstrate that when used in combination with a single, traditional soft lithography process, it is possible to generate hierarchically-organized membrane domains and microscale 2-D array patterns of domains. Significantly, this technique can be used to repeatedly modify membranes allowing iterative control over membrane composition. This approach expands our toolkit for functional membrane design, with potential applications for enhanced materials templating, biosensing and investigating lipid-membrane processes.

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na(+) leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca(2+) gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Understanding the pathophysiology of tuberculosis, and the bio-distribution of pathogen-associated molecules in the host is essential for the development of efficient methods of intervention. One of the key virulence factors in the pathology of tuberculosis infection is Lipoarabinomannan (LAM). Previously, we have demonstrated the reliable detection of LAM in urine from tuberculosis patients in a sandwich immunoassay format. We have also applied an ultra-sensitive detection strategy developed for amphiphilic biomarkers, membrane insertion, to the detection of LAM with a limit of detection of 10 fM. Herein, we evaluate the application of membrane insertion to the detection of LAM in patient serum, and demonstrate that the circulating concentrations of 'monomeric' LAM in serum are very low, despite significantly higher concentrations in the urine. Using spiked samples, we demonstrate that this discrepancy is due to the association of LAM with high-density lipoprotein (HDL) nanodiscs in human serum. Indeed, pull-down of HDL nanodiscs from human serum allows for the recovery of HDL-associated LAM. These studies suggest that LAM is likely associated with carrier molecules such as HDL in the blood of patients infected with tuberculosis. This phenomenon may not be limited to LAM in that many pathogen-associated molecular patterns like LAM are amphiphilic in nature and may also be associated with host lipid carriers. Such interactions are likely to affect host-pathogen interactions, pathogen bio-distribution and clearance in the host, and must be thoroughly understood for the effective design of vaccines and diagnostics., (Published by Elsevier Ltd.)

Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high non-specific interactions are used for the detection of such biomarkers. In this manuscript, we demonstrate the development of sandwich immunoassays for the direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions allows for the ultra-sensitive and quantitative detection of biomarkers (an order of magnitude enhanced sensitivity, as compared to plate-based ELISA) in complex patient samples (urine, serum) within a short time. We also demonstrate the detection of LAM in urine from a small sample of subjects being treated for TB using this approach with excellent sensitivity and 100% corroboration with disease status. These results suggest that pathogen-specific biomarkers can be applied for the rapid and effective diagnosis of disease. It is likely that detection of a combination of biomarkers offers greater reliability of diagnosis, rather than detection of any single pathogen biomarker. NCT00341601., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)