Mitochondrial respiratory-chain adaptations in macrophages contribute to antibacterial host defense.

Macrophages tightly scale their core metabolism after being activated, but the precise regulation of the mitochondrial electron-transport chain (ETC) and its functional implications are currently unknown. Here we found that recognition of live bacteria by macrophages transiently decreased assembly of the ETC complex I (CI) and CI-containing super-complexes and switched the relative contributions of CI and CII to mitochondrial respiration. This was mediated by phagosomal NADPH oxidase and the reactive oxygen species (ROS)-dependent tyrosine kinase Fgr. It required Toll-like receptor signaling and the NLRP3 inflammasome, which were both connected to bacterial viability-specific immune responses. Inhibition of CII during infection with Escherichia coli normalized serum concentrations of interleukin 1ß (IL-1ß) and IL-10 to those in mice treated with dead bacteria and impaired control of bacteria. We have thus identified ETC adaptations as an early immunological-metabolic checkpoint that adjusts innate immune responses to bacterial infection.

Conformational Control of Cascade Interference and Priming Activities in CRISPR Immunity.

During type I-E CRISPR-Cas immunity, the Cascade surveillance complex utilizes CRISPR-derived RNAs to target complementary invasive DNA for destruction. When invader mutation blocks this interference activity, Cascade instead triggers rapid primed adaptation against the invader. The molecular basis for this dual Cascade activity is poorly understood. Here we show that the conformation of the Cse1 subunit controls Cascade activity. Using FRET, we find that Cse1 exists in a dynamic equilibrium between "open" and "closed" conformations, and the extent to which the open conformation is favored directly correlates with the attenuation of interference and relative increase in priming activity upon target mutation. Additionally, the Cse1 L1 motif modulates Cascade activity by stabilizing the closed conformation. L1 mutations promote the open conformation and switch immune response from interference to priming. Our results demonstrate that Cascade conformation controls the functional outcome of target recognition, enabling tunable CRISPR immune response to combat invader evolution.

The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity.

All organisms need to continuously adapt to changes in their environment. Through horizontal gene transfer, bacteria and archaea can rapidly acquire new traits that may contribute to their survival. However, because new DNA may also cause damage, removal of imported DNA and protection against selfish invading DNA elements are also important. Hence, there should be a delicate balance between DNA uptake and DNA degradation. Here, we describe prokaryotic antiviral defense systems, such as receptor masking or mutagenesis, blocking of phage DNA injection, restriction/modification, and abortive infection. The main focus of this review is on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated), a prokaryotic adaptive immune system. Since its recent discovery, our biochemical understanding of this defense system has made a major leap forward. Three highly diverse CRISPR/Cas types exist that display major structural and functional differences in their mode of generating resistance against invading nucleic acids. Because several excellent recent reviews cover all CRISPR subtypes, we mainly focus on a detailed description of the type I-E CRISPR/Cas system of the model bacterium Escherichia coli K12.

CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3.

The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.

Aging-Impaired Filamentous Actin Polymerization Signaling Reduces Alveolar Macrophage Phagocytosis of Bacteria.

In elderly patients, bacterial infection often causes severe complications and sepsis. Compared to younger patients, older patients are more susceptible to sepsis caused by respiratory infection. Macrophage (Mϕ) phagocytosis of bacteria plays a critical role in the clearance of pathogens and the initiation of immune responses. It has been suggested that Mϕ exhibit age-related functional alterations, including reduced chemotaxis, phagocytosis, antibacterial defense, and the ability to generate reactive oxygen species. However, the mechanisms behind these changes remain unclear. The present study sought to determine changes in bacterial phagocytosis in aging alveolar Mϕ (AMϕ) and the underlying mechanisms. We show that bacteria initiate cytoskeleton remodeling in AMϕ through interaction with macrophage receptor with collagenous structure (MARCO), a bacterial scavenger receptor. This remodeling, in turn, promotes enhanced cell surface expression of MARCO and bacterial phagocytosis. We further demonstrate that Rac1-GTP mediates MARCO signaling and activates actin-related protein-2/3 complex, an F-actin nucleator, thereby inducing F-actin polymerization, filopodia formation, and increased cell surface expression of MARCO, all of which are essential for the execution of bacteria phagocytosis. However, AMϕ isolated from aging mice exhibit suppressed Rac1 mRNA and protein expression, which resulted in decreases in Rac1-GTP levels and actin-related protein-2/3 activation, as well as subsequent attenuation of F-actin polymerization, filopodia formation, and cell surface expression of MARCO. As a result, bacterial phagocytosis in aging AMϕ is decreased. This study highlights a previously unidentified mechanism by which aging impairs Mϕ phagocytosis of bacteria. Targeting these pathways may improve outcomes of bacterial infection in elderly patients.

Histone deacetylase inhibition enhances antimicrobial peptide but not inflammatory cytokine expression upon bacterial challenge.

Antimicrobial peptides (AMP) are defense effectors of the innate immunity playing a crucial role in the intestinal homeostasis with commensals and protection against pathogens. Herein we aimed to investigate AMP gene regulation by deciphering specific characteristics allowing their enhanced expression among innate immune genes, particularly those encoding proinflammatory mediators. Our emphasis was on epigenetic regulation of the gene encoding the AMP ß-defensin 2 (HBD2), taken as a model of possibly specific induction, upon challenge with a commensal bacterium, compared with the proinflammatory cytokine IL-8. Using an in vitro model of colonic epithelial cells challenged with Escherichia coli K12, we showed that inhibition of histone deacetylases (HDAC) by trichostatin A dramatically enhanced induction of HBD2 expression, without affecting expression of IL-8. This mechanism was supported by an increased phosphorylation of histone H3 on serine S10, preferentially at the HBD2 promoter. This process occurred through activation of the IκB kinase complex, which also led to activation of NF-κB. Moreover, we demonstrated that NF-κB was modified by acetylation upon HDAC inhibition, partly by the histone acetyltransferase p300, and that both NF-κB and p300 supported enhanced induction of HBD2 expression. Furthermore, we identified additional genes belonging to antimicrobial defense and epithelial restitution pathways that showed a similar pattern of epigenetic control. Finally, we confirmed our finding in human colonic primary cells using an ex vivo organoid model. This work opens the way to use epigenetic pharmacology to achieve induction of epithelial antimicrobial defenses, while limiting the deleterious risk of an inflammatory response.

Structures of the RNA-guided surveillance complex from a bacterial immune system.

Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5' end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences.

Degenerate target sites mediate rapid primed CRISPR adaptation.

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed "priming." Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.

CD16 promotes Escherichia coli sepsis through an FcR gamma inhibitory pathway that prevents phagocytosis and facilitates inflammation.

Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here we show that the FcRgamma adaptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing signal transduction subunit of the Fc receptor family, has a deleterious effect on sepsis. FcRgamma(-/-) mice show increased survival during peritonitis, owing to markedly increased E. coli phagocytosis and killing and to lower production of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The FcRgamma-associated receptor that inhibits E. coli phagocytosis is FcgammaRIII (also called CD16), and its absence protects mice from sepsis. FcgammaRIII binds E. coli, and this interaction induces FcRgamma phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibits E. coli phagocytosis and increases TNF-alpha production through Toll-like receptor 4. We identified the phagocytic receptor negatively regulated by FcRgamma on macrophages as the class A scavenger receptor MARCO. E. coli-FcgammaRIII interaction induces the recruitment of SHP-1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding FcgammaRIII, E. coli triggers an inhibitory FcRgamma pathway that both impairs MARCO-mediated bacterial clearance and activates TNF-alpha secretion.

Surface functionalization of thin-film diamond for highly stable and selective biological interfaces.

Carbon is an extremely versatile family of materials with a wide range of mechanical, optical, and mechanical properties, but many similarities in surface chemistry. As one of the most chemically stable materials known, carbon provides an outstanding platform for the development of highly tunable molecular and biomolecular interfaces. Photochemical grafting of alkenes has emerged as an attractive method for functionalizing surfaces of diamond, but many aspects of the surface chemistry and impact on biological recognition processes remain unexplored. Here we report investigations of the interaction of functionalized diamond surfaces with proteins and biological cells using X-ray photoelectron spectroscopy (XPS), atomic force microscopy, and fluorescence methods. XPS data show that functionalization of diamond with short ethylene glycol oligomers reduces the nonspecific binding of fibrinogen below the detection limit of XPS, estimated as > 97% reduction over H-terminated diamond. Measurements of different forms of diamond with different roughness are used to explore the influence of roughness on nonspecific binding onto H-terminated and ethylene glycol (EG)-terminated surfaces. Finally, we use XPS to characterize the chemical stability of Escherichia coli K12 antibodies on the surfaces of diamond and amine-functionalized glass. Our results show that antibody-modified diamond surfaces exhibit increased stability in XPS and that this is accompanied by retention of biological activity in cell-capture measurements. Our results demonstrate that surface chemistry on diamond and other carbon-based materials provides an excellent platform for biomolecular interfaces with high stability and high selectivity.

CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids.

Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid's mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences.

Monocyte-mediated inhibition of TLR9-dependent IFN-α induction in plasmacytoid dendritic cells questions bacterial DNA as the active ingredient of bacterial lysates.

Bacterial DNA contains unmethylated CpG dinucleotides and is a potent ligand for TLR9. Bacterial DNA has been claimed the active ingredient in bacterial lysates used for immunotherapy. Whereas the detection of viral DNA by TLR9 expressed in plasmacytoid dendritic cells (PDCs) with subsequent IFN-α production is well defined, the role of bacterial DNA during microbial infection is less clear. In fact, IFN-α is not a hallmark of antibacterial immune responses. Unlike in mice, TLR9 expression in humans is restricted to PDCs and B cells; thus, conclusions from murine models of infection have limitations. In this study, we demonstrate that lysates of heat-killed Escherichia coli containing bacterial DNA induced IFN-α in isolated PDCs but not in the mixed cell populations of human PBMCs. Depletion of monocytes restored IFN-α secretion by PDCs within PBMCs. We found that monocyte-derived IL-10 and PGs contribute to monocyte-mediated inhibition of IFN-α release in PDCs. We conclude that human PDCs can be stimulated by bacterial DNA via TLR9; however, in the physiological context of mixed-cell populations, PDC activation is blocked by factors released from monocytes stimulated in parallel by other components of bacterial lysates such as LPS. This functional repression of PDCs by concomitantly stimulated monocytes avoids production of antiviral IFN-α during bacterial infection and thus explains how the innate immune system is enabled to distinguish bacterial from viral CpG DNA and thus to elicit the appropriate responses despite the presence of CpG DNA in both types of infection.

Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae.

Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae.

Functional roles of mannose-binding protein in the adhesion, cytotoxicity and phagocytosis of Acanthamoeba castellanii.

Acanthamoeba castellanii is a single-celled protozoan that is widely distributed in the environment and is a well-known of causing human keratitis, a vision-threatening infection. In this study, an ethyl methane sulfonate (EMS) and a selection of saccharide were applied to A. castellanii by chemical mutagenesis. To understand the functional roles of a mannose-binding protein (MBP). A. castellanii were treated with methyl-alpha-D-mannopyranoside abbreviated Man, with and without the EMS pre-treatment, and their adhesion and cytotoxicity were analyzed, using a human brain microvascular endothelial cell (HBMEC) as the target cell. Both EMS and Man mutants exhibited significantly decreased levels of MBP expression and cytotoxicity to HBMEC, but showed similar levels of binding to HBMEC, as compared with the wild type. Of interest was that the exogenous mannose inhibited amoebae (i.e., Man mutant) binding to the HBMEC by <20%. Only the mutant Man exhibited a significant decrease in bacterial uptake, as compared to the wild type, 0.020 vs 0.032 (p<0.05) and proteolytic activity. The results showed that MBP should be clearly provided as the pathogenic target candidate, to further target-based therapy, but EMS mutation should not be associated with initial adhesion and phagocytosis of A. castellanii.

Escherichia coli flagellin stimulates pro-inflammatory immune response.

Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system. Recognition of flagellin by innate immune receptors stimulates the production of cytokines necessary for the development of effective immunity. Here, we demonstrated that the intranasal (i.n.) instillation of different amount of Escherichia coli K-12 flagellin preparation (0.5, 1, 2, 4 µg) in BALB/c mice induced pro-inflammatory immune response. Instillation i.n. of 1 µg of flagellin induced the maximum expression of interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) mRNA and production of pro-inflammatory cytokines (IL-1ß, TNF-α and IL-6) in mice lungs. The same dose of flagellin induced neutrophil polymorphonuclear cells infiltration in peribronchial and perivascular regions. High number of neutrophil in bronchoalveolar lavage fluid was found at 24 h after i.n. instillation of flagellin (1 µg). These findings were concomitant with the maximum production of myeloperoxidase and nitric oxide in mice lungs. Present study showed that the maximum pro-inflammatory mediator levels were found when mice instilled i.n. with 1 µg E. coli flagellin. The amount of flagellin of E. coli K-12 that achieve the maximum stimulation of mucosal pro-inflammatory immune response in mice lungs was explored in this study.

H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO.

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

Engineering nanostructured porous SiO2 surfaces for bacteria detection via "direct cell capture".

An optical label-free biosensing platform for bacteria detection ( Escherichia coli K12 as a model system) based on nanostructured oxidized porous silicon (PSiO(2)) is introduced. The biosensor is designed to directly capture the target bacteria cells on its surface with no prior sample processing (such as cell lysis). The optical reflectivity spectrum of the PSiO(2) nanostructure displays Fabry-Pérot fringes characteristic of thin-film interference, enabling direct, real-time observation of bacteria attachment within minutes. The PSiO(2) optical nanostructure is synthesized and used as the optical transducer element. The porous surface is conjugated with specific monoclonal antibodies (immunoglobulin G's) to provide the active component of the biosensor. The immobilization of the antibodies onto the biosensor system is confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, fluorescent labeling experiments, and refractive interferometric Fourier transform spectroscopy. We show that the immobilized antibodies maintain their immunoactivity and specificity when attached to the sensor surface. Exposure of these nanostructures to the target bacteria results in "direct cell capture" onto the biosensor surface. These specific binding events induce predictable changes in the thin-film optical interference spectrum of the biosensor. Our preliminary studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations. The current detection limit of E. coli K12 bacteria is 10(4) cells/mL within several minutes.

The real-time-based assessment of the microbial killing by the antimicrobial compounds of neutrophils.

A recombinant Escherichia coli K-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE) from Photorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. This E. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL), which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H(2)O(2)) and myeloperoxidase (MPO) was assessed. In high concentrations, H(2)O(2) alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl). Taurine, the known HOCl scavenger, blocked the killing by MPO. When E. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H(2)O(2)/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

Regulation of IL-8 production by complement-activated product, C5a, in vitro and in vivo during sepsis.

Excessive complement-activated product complement 5a (C5a) has been implicated in the pathogenesis of sepsis development. Herein, we employed in vitro and in vivo models of sepsis to investigate the functional relationship between overtly produced C5a and IL-8. Our data revealed that C5a could strongly amplify IL-8 expression from human whole blood cells induced by LPS and other types of TLR agonists. ERK1/2 and p38, but not JNK, were mainly participated in signaling pathways for IL-8 production. In the whole blood stimulated by Escherichiacoli, C5a levels were quickly elevated and blockage of C5a significantly decreased E. coli-elicited IL-8 production. In the mouse model of sepsis induced by cecal ligation and puncture (CLP), the markedly increased keratinocyte-derived cytokine (KC) could be strongly suppressed by blockage of C5a. These data suggest that excessive C5a functions as a critical inflammatory mediator to enhance IL-8 production mainly through MAPK signaling pathways.

Tunable two-dimensional array patterning of antibody annuli through microsphere templating.

Protein microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is a lack of techniques that can result in annulus-shaped protein structures (e.g., for the utilization of curved surfaces for enhanced protein-protein interactions and the detection of antigens). We present a new technique employing colloidal templating to yield large-scale (approximately cm(2)) 2D arrays of antibodies against Escherichia coli K12 and enhanced green fluorescent protein (eGFP) on a versatile glass surface. The antibodies are swept to reside around the templating microspheres during solution drying and physically adsorb onto the glass. After the microspheres are removed, an array of annulus-shaped antibody structures is formed. We demonstrate the preserved antibody structure and functionality by binding the specific antigens and secondary antibodies, respectively, which paves the way for the binding of biomolecules and pathogens such as bacteria and viruses. The structures were investigated via atomic force, confocal, and fluorescence microscopy. Operational factors such as the drying time, temperature, and humidity as well as the presence of surfactants in the antibody solution were tuned to obtain a stable antibody structure.