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Biomimetic Virulomics for Capture and Identification of Cell-Type Specific Effector Proteins John D Lapek, Jr., Ronnie H Fang, Xiaoli Wei, Pengyang Li, Bo Wang, Liangfang Zhang, and David J Gonzalez ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b02650 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017
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Biomimetic Virulomics for Capture and Identification of Cell-Type Specific Effector Proteins John D. Lapek, Jr.1,2, Ronnie H. Fang3,4, Xiaoli Wei3,4, Pengyang Li5, Bo Wang5*, Liangfang Zhang3,4*, David J. Gonzalez1,2* 1
Department of Pharmacology, 2Skaggs School of Pharmacy and Pharmaceutical Sciences,
3
Department of NanoEngineering, 4Moores Cancer Center, University of California, San Diego,
La Jolla, CA, USA, 5Department of Bioengineering, Stanford University, Stanford, CA, USA.
KEYWORDS biomimetics, affinity enrichment, quantitative proteomics, mass spectrometry, virulence factors
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ABSTRACT An unmet challenge in the study of disease is to accurately streamline the identification of important virulence factors. Traditional, genetically driven approaches miss biologically relevant markers due to discordance between the genome and proteome. Here, we developed a nanotechnology-enabled affinity enrichment strategy coupled with multiplexed quantitative proteomics, namely Biomimetic Virulomics, for successful identification of cell-type specific effector proteins of both prokaryotic and eukaryotic pathogens. We highlight the power of Biomimetic Virulomics by capturing known virulence factors in a high throughput, cell type guided fashion. Additionally, a comprehensive characterization of the membrane protein component of biomimetics utilized in this strategy is provided. Interfacing cell-derived nanomaterials with multiplexed quantitative proteomics allows for a specific targeting strategy of virulence factors that can be utilized for drug discovery against prominent human diseases.
The term virulence is commonly used regarding infectious disease. By its definition however, virulence can be used to describe the severity of any disease. As such, virulence factors can be defined as molecules that add to the effectiveness of a disease, and thus allow the disease to thrive in a host. Diseases that can be encompassed by this definition not only include those of bacterial or viral origin, but also a plethora of diseases including cancer.1 Conversely, host-derived defense molecules can also be included under the umbrella of virulence factors, as they are molecules that aid in the prosperity of the parent organism in its environment while eliciting a virulent effect on their target. Under a classical definition of virulence, infectious
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diseases remain a major global health concern with associated high morbidity and mortality rates.2 Within host-pathogen interactions, virulence is governed by biomolecules produced by the pathogen that target different tissues during infection. Thus, the identification of pathogenic effector proteins with cell-type specificity is paramount in understanding pathophysiology associated with infection and for future development of effective intervention strategies. However, the identification of relevant virulence factors, particularly at the protein-level, has proven to be challenging. Historically, direct detection of virulence-associated proteins by mass spectrometry has been hindered by high background from host proteins, making a proteomics approach difficult.3 Biomimetic nanosponges (NS), consisting of polymeric nanoparticle cores coated with endogenous cell membranes, have emerged as an effective means of neutralizing and retaining pore forming toxins4 - a classical virulence mechanism. NS are currently available for a variety of host cell types,5-8 and have even been coated with bacterial membranes,9 thus demonstrating the potential for broad application of this technology. The endogenous membrane coatings camouflage the NS, allowing them to act as decoys for virulence factors that target specific host cell types. However, previous studies utilizing NS do not identify the virulence factors they neutralize a priori. Biomimetics have the added advantage of avoiding destruction by the host immune system.10-11 Intriguingly, biomimetics are known to bind and neutralize toxins while maintaining natural conformations of toxin proteins, allowing the immune system to mount a response and increase survival.6
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Through this study, we sought to overcome the challenges associated with direct identification of virulence factors, biochemically understand the constituents that comprise a NS, and identify proteins bound and neutralized by the NS. Towards this goal, multiplexed quantitative proteomics12 was combined with cell-type specific NS affinity capture to yield an affinity enrichment workflow, Biomimetic Virulomics (Figure 1). Multiplexed quantitative proteomics gives a two-fold advantage over normal proteomics. First, the issue of missing values between replicates and samples can be addressed, while increasing throughput.13 In a traditional proteomics experiment missing values can lead to false positives and false negatives, where proteins may be considered true or false binders based solely on their presence or absence in a list of identified proteins. Second, the relative quantitation enabled by multiplexed quantitative proteomics allows us to determine fold-enrichment, a measure of specificity, when comparing samples. RESULTS/DISCUSSION Prior to applying the workflow, we first characterized the NS used in subsequent experiments. NS with red blood cell (RBC) membrane (denoted RBCNS) and macrophage membrane (denoted MNS) were prepared for host cell-type specific enrichment of pathogenderived effector proteins (Figure 2). For both RBCNS and MNS, an increase in diameter was observed once the NS underwent the membrane cloaking process (Figure 2a and 2d). Furthermore, a reduction in zeta potential, indicating the presence of the membrane and its insulating properties (Figure 2b and 2e), was noted. Membrane coating, known to exist in a right-side-out orientation,14 was confirmed by electron microscopy, and the particles exhibited a characteristic core-shell structure (Figure 2c and 2f). NS made from both RBC and nucleated
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cell membranes have been previously shown to possess narrow size distributions and exceptional stability under physiological conditions.5, 11 Having confirmed the presence of membranes on the NS, we sought to define the hostcell type derived protein component of the membranes cloaking the NS. The rationale for this is that virulence factors can bind, inhibit and even degrade specific host membrane proteins through a variety of mechanisms.15-16 Understanding which host membrane proteins are present allows us to leverage this information to guide future mechanistic studies of virulence factor action. Quantitative proteomics was used to characterize two host-derived RBCNS, human and mouse, and MNS from mice (Figure 3). In total, 981 proteins were identified specifically associated with the human RBCNS. Of these, 937 (>95%) were statistically specific, assessed by π value,17 to the RBCNS (Figure 3a). The π value was used as a statistical measure to account for variance in measurement due to the heterogeneity of the RBCNS population. The remaining proteins were likely not statistically associated due to degradation of a small population of RBCNS or from residual membrane component in the supernatant from the coating process. Though these results are not a comprehensive catalogue of all RBC proteins, they represent an advance in the number of proteins derived from RBCs,18 where previous studies required a compendium of techniques to define only 592 RBC proteins. Additionally, there is well-documented difficulty in proteomics relating to the identification of membrane proteins, especially multi-pass transmembrane proteins.19-21 Taken together with previous in vivo and in vitro findings,4-11 RBCNS demonstrate an ability to accurately reflect interactions between host cells and pathogens. Despite this, it is possible that not every host cell-pathogen interaction will be captured or identified, regardless of the cell type. Failure to identify interactions could arise
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from technical limitations of the mass spectrometer, the length and nature of the effector molecule, and whether or not the effector molecule embeds in the membrane with high affinity. To demonstrate that the proteins on the RBCNS are derived from RBCs, we show a 77fold enrichment of the erythrocyte membrane protein BAND322 relative to the supernatant (Figure 3b); BAND3 constitutes up to 25% of the membrane protein component of RBCs. Additionally, the majority of the highly abundant RBC proteins22 (Band3, Band2, Protein 4.2, Ankyrin, beta-spectrin, XK, Glut1, Dematin, Adducin, Tropomyosin and Tropomodulin) were present and enriched in the RBCNS fraction (Supplementary Table 1). This indicates that the complex membrane network is preserved on the RBCNS. For mouse-derived RBCNS and MNS, the samples were directly compared at the proteome level. This provided the opportunity to determine not only the protein component of the nanosponges derived from the host, but also define cell-type specific proteins. In total, 5,580 mouse proteins were determined to comprise the host component of the NS. Of these, 1,220 were specific to the MNS and 70 were significantly enriched on the RBCNS (Figure 3c). These results confirm the relative complexity of a macrophage relative to an RBC. Markers and enrichment of RBCNS from mouse agreed with those from the described human derived RBCNS (Supplementary Figure 1). Specific markers for macrophages, including Emr1, CD68, Csf1r and Lgals323 were enriched on MNS compared to RBCNS. A full list of identified proteins and quantitation values is available in Supplementary Table 1. The results from the characterization of the protein component of the NS show a drastic reduction in host derived proteins, as a typical proteomics experiment in hematopoietic cells would identify upwards of 10,000 proteins.24 We hypothesized that this reduction in complexity
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of host-derived sample matrix would allow a deeper characterization of proteins derived from pathogens with full implementation of the Biomimetic Virulomics workflow. To benchmark the Biomimetic Virulomics platform, we utilized human-derived RBCNS to selectively enrich for hemolytic proteins from Group A streptococcus (GAS), a humanspecific pathogen. As hemolysis is a characteristic virulence mechanism of GAS and a clinical diagnostic phenotype, we first confirmed the ability of the RBCNS to bind and neutralize the hemolytic activity of secreted proteins collected from GAS cultures, recapitulating previous work.4 Cell-free supernatants from GAS were fractionated to identify hemolytic fractions (HF),25 and lytic ability was confirmed by heme release from blood. After incubation of the HF with the RBCNS, no quantifiable lysis was observed, indicating neutralization of the hemolytic proteins (Figure 4a). Upon confirming neutralization of lysis, RBCNS were incubated with cell-free GAS supernatants to identified GAS proteins specifically bound to the RBCNS. In parallel, the effect of blocking RBCNS was assessed with bovine serum albumin prior to incubation with GAS supernatants. A statistical difference in specific binding proteins was not observed (Supplementary Figures 2, 3). As such, blocked NS were utilized for subsequent characterization with the assumption that blocking more accurately mimicked in vivo conditions. Prior to full implementation, we ensured blocking did not affect the stability of proteins associated with the nanosponges (Supplementary Figures 4, 5). In total, 602 GAS proteins were identified (35% of the GAS proteome), with only 58 (
