An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells.

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

Galectin-8 Downmodulates TLR4 Activation and Impairs Bacterial Clearance in a Mouse Model of Pseudomonas aeruginosa Keratitis.

Pseudomonas aeruginosa provokes a painful, sight-threatening corneal infection. It progresses rapidly and is difficult to treat. In this study, using a mouse model of P. aeruginosa keratitis, we demonstrate the importance of a carbohydrate-binding protein, galectin-8 (Gal-8), in regulation of the innate immune response. First, using two distinct strains of P. aeruginosa, we established that Gal-8-/- mice are resistant to P. aeruginosa keratitis. In contrast, mice deficient in Gal-1, Gal-3, and Gal-9 were fully susceptible. Second, the addition of exogenous rGal-8 to LPS (TLR4 ligand)-stimulated bone marrow-derived macrophages suppressed 1) the activation of the NF-κB pathway, and 2) formation of the MD-2/TLR4 complex. Additionally, the expression level of reactive oxygen species was substantially higher in infected Gal-8-/- bone marrow neutrophils (BMNs) compared with the Gal-8+/+ BMNs, and the P. aeruginosa killing capacity of Gal-8-/- BMNs was considerably higher compared with that of Gal-8+/+ BMNs. In the bacterial killing assays, the addition of exogenous rGal-8 almost completely rescued the phenotype of Gal-8-/- BMNs. Finally, we demonstrate that a subconjunctival injection of a Gal-8 inhibitor markedly reduces the severity of infection in the mouse model of P. aeruginosa keratitis. These data lead us to conclude that Gal-8 downmodulates the innate immune response by suppressing activation of the TLR4 pathway and clearance of P. aeruginosa by neutrophils. These findings have broad implications for developing novel therapeutic strategies for treatment of conditions resulting from the hyperactive immune response both in ocular as well as nonocular tissues.

Pseudomonas aeruginosa-induced nociceptor activation increases susceptibility to infection.

We report a rapid reduction in blink reflexes during in vivo ocular Pseudomonas aeruginosa infection, which is commonly attributed and indicative of functional neuronal damage. Sensory neurons derived in vitro from trigeminal ganglia (TG) were able to directly respond to P. aeruginosa but reacted significantly less to strains of P. aeruginosa that lacked virulence factors such as pili, flagella, or a type III secretion system. These observations led us to explore the impact of neurons on the host's susceptibility to P. aeruginosa keratitis. Mice were treated with Resiniferatoxin (RTX), a potent activator of Transient Receptor Potential Vanilloid 1 (TRPV1) channels, which significantly ablated corneal sensory neurons, exhibited delayed disease progression that was exemplified with decreased bacterial corneal burdens and altered neutrophil trafficking. Sensitization to disease was due to the increased frequencies of CGRP-induced ICAM-1+ neutrophils in the infected corneas and reduced neutrophil bactericidal activities. These data showed that sensory neurons regulate corneal neutrophil responses in a tissue-specific matter affecting disease progression during P. aeruginosa keratitis. Hence, therapeutic modalities that control nociception could beneficially impact anti-infective therapy.

Microglia and Neutrophils to the Rescue.

Invasive candidiasis is a medical condition that is challenging to treat. A recent study (Drummond et al.Nat. Immunol. 2019;20:559-570) reveals new insights into the role of microglia and neutrophils in limiting fungal dissemination to the mouse brain. Gaining knowledge into tissue-specific protection mechanisms may ensure improved prognosis and disease management against fungal infections.

Cochlin produced by follicular dendritic cells promotes antibacterial innate immunity.

Cochlin, an extracellular matrix protein, shares homologies with the Factor C, a serine protease found in horseshoe crabs, which is critical for antibacterial responses. Mutations in the COCH gene are responsible for human DFNA9 syndrome, a disorder characterized by neurodegeneration of the inner ear that leads to hearing loss and vestibular impairments. The physiological function of cochlin, however, is unknown. Here, we report that cochlin is specifically expressed by follicular dendritic cells and selectively localized in the fine extracellular network of conduits in the spleen and lymph nodes. During inflammation, cochlin was cleaved by aggrecanases and secreted into blood circulation. In models of lung infection with Pseudomonas aeruginosa and Staphylococcus aureus, Coch(-/-) mice show reduced survival linked to defects in local cytokine production, recruitment of immune effector cells, and bacterial clearance. By producing cochlin, FDCs thus contribute to the innate immune response in defense against bacteria.

Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface.

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

Impact of Microbiota on Resistance to Ocular Pseudomonas aeruginosa-Induced Keratitis.

The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking. We compared the relative contribution of eye and gut commensals in regulating the ocular susceptibility to Pseudomonas aeruginosa-induced keratitis. We find that in health, the presence of microbiota strengthened the ocular innate immune barrier by significantly increasing the concentrations of immune effectors in the tear film, including secretory IgA and complement proteins. Consistent with this view, Swiss Webster (SW) mice that are typically resistant to P. aeruginosa-induced keratitis become susceptible due to the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the germ free (GF) mice when compared to the conventionally maintained SW mice. The protective immunity was found to be dependent on both eye and gut microbiota with the eye microbiota having a moderate, but significant impact on the resistance to infection. These events were IL-1ß-dependent as corneal IL-1ß levels were decreased in the infected GF and antibiotic-treated mice when compared to the SPF controls, and neutralization of IL-1ß increased the ocular bacterial burden in the SPF mice. Monocolonizing GF mice with Coagulase Negative Staphylococcus sp. isolated from the conjunctival swabs was sufficient to restore resistance to infection. Cumulatively, these data underline a previously unappreciated role for microbiota in regulating susceptibility to ocular keratitis. We predict that these results will have significant implications for contact lens wearers, where alterations in the ocular commensal communities may render the ocular surface vulnerable to infections.

Immune Recognition of the Epidemic Cystic Fibrosis Pathogen Burkholderia dolosa.

Burkholderia dolosa caused an outbreak in the cystic fibrosis (CF) clinic at Boston Children's Hospital from 1998 to 2005 and led to the infection of over 40 patients, many of whom died due to complications from infection by this organism. To assess whether B. dolosa significantly contributes to disease or is recognized by the host immune response, mice were infected with a sequenced outbreak B. dolosa strain, AU0158, and responses were compared to those to the well-studied CF pathogen Pseudomonas aeruginosa In parallel, mice were also infected with a polar flagellin mutant of B. dolosa to examine the role of flagella in B. dolosa lung colonization. The results showed a higher persistence in the host by B. dolosa strains, and yet, neutrophil recruitment and cytokine production were lower than those with P. aeruginosa The ability of host immune cells to recognize B. dolosa was then assessed, B. dolosa induced a robust cytokine response in cultured cells, and this effect was dependent on the flagella only when bacteria were dead. Together, these results suggest that B. dolosa can be recognized by host cells in vitro but may avoid or suppress the host immune response in vivo through unknown mechanisms. B. dolosa was then compared to other Burkholderia species and found to induce similar levels of cytokine production despite being internalized by macrophages more than Burkholderia cenocepacia strains. These data suggest that B. dolosa AU0158 may act differently with host cells and is recognized differently by immune systems than are other Burkholderia strains or species.

Intestinal Microbiota of Mice Influences Resistance to Staphylococcus aureus Pneumonia.

Th17 immunity in the gastrointestinal tract is regulated by the intestinal microbiota composition, particularly the presence of segmented filamentous bacteria (sfb), but the role of the intestinal microbiota in pulmonary host defense is not well explored. We tested whether altering the gut microbiota by acquiring sfb influences the susceptibility to staphylococcal pneumonia via induction of type 17 immunity. Groups of C57BL/6 mice which differed in their intestinal colonization with sfb were challenged with methicillin-resistant Staphylococcus aureus in an acute lung infection model. Bacterial burdens, bronchoalveolar lavage fluid (BALF) cell counts, cell types, and cytokine levels were compared between mice from different vendors, mice from both vendors after cohousing, mice given sfb orally prior to infection, and mice with and without exogenous interleukin-22 (IL-22) or anti-IL-22 antibodies. Mice lacking sfb developed more severe S. aureus pneumonia than mice colonized with sfb, as indicated by higher bacterial burdens in the lungs, lung inflammation, and mortality. This difference was reduced when sfb-negative mice acquired sfb in their gut microbiota through cohousing with sfb-positive mice or when given sfb orally. Levels of type 17 immune effectors in the lung were higher after infection in sfb-positive mice and increased in sfb-negative mice after acquisition of sfb, as demonstrated by higher levels of IL-22 and larger numbers of IL-22(+) TCRß(+) cells and neutrophils in BALF. Exogenous IL-22 protected mice from S. aureus pneumonia. The murine gut microbiota, particularly the presence of sfb, promotes pulmonary type 17 immunity and resistance to S. aureus pneumonia, and IL-22 protects against severe pulmonary staphylococcal infection.

Homotrimeric macrophage migration inhibitory factor (MIF) drives inflammatory responses in the corneal epithelium by promoting caveolin-rich platform assembly in response to infection.

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa-induced IL-8 responses via the formation of caveolin-1-rich "signaling hubs" in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.

Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity.

Ocular bacterial keratitis, often associated with Pseudomonas aeruginosa bacterial infection, commonly occurs in contact lens wearers and may lead to vision impairment. In this study, we analyzed the contribution of neutrophil extracellular traps (NETs) to the mediation of protection during ocular keratitis. Both invasive and cytotoxic P. aeruginosa clinical isolates induced NET release by neutrophils. NETs carried the characteristic histone proteins, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the invasive P. aeruginosa strains PAO1 (serogroup O5) and 6294 (serogroup O6) were trapped by NETs, the cytotoxic P. aeruginosa strains 6077, 6206 (serogroup O11), and PA14 (serogroup 010) were less sensitive to NET capture. The mechanism of escape by the cytotoxic strains from adhesion to NETs involved the shedding of outer membrane vesicles (OMVs) that outcompeted the cytotoxic P. aeruginosa strains for NET binding. When ocular infection was caused by an invasive strain in vivo, NETs were released at the ocular surface to capture bacteria, limiting their spread. Treatment with MNase I had a dose-dependent effect, with low doses of MNase speeding up bacterial clearance and high doses of MNase having toxic consequences. Cumulatively, our data suggest that NET-mediated immunity is a two-step process. Initially, pathogens attach to NET fragments; subsequently, upon nuclease activity, active serine proteases, which proteolytically degrade NET-associated proteins and promote DNase activity, are released. Therefore, a balance between NET production and NET degradation is needed to achieve maximal NET immunity.

mtx-COBRA: Subcellular localization prediction for bacterial proteins.

BACKGROUND: Bacteria can have beneficial effects on our health and environment; however, many are responsible for serious infectious diseases, warranting the need for vaccines against such pathogens. Bioinformatic and experimental technologies are crucial for the development of vaccines. The vaccine design pipeline requires identification of bacteria-specific antigens that can be recognized and can induce a response by the immune system upon infection. Immune system recognition is influenced by the location of a protein. Methods have been developed to determine the subcellular localization (SCL) of proteins in prokaryotes and eukaryotes. Bioinformatic tools such as PSORTb can be employed to determine SCL of proteins, which would be tedious to perform experimentally. Unfortunately, PSORTb often predicts many proteins as having an "Unknown" SCL, reducing the number of antigens to evaluate as potential vaccine targets. METHOD: We present a new pipeline called subCellular lOcalization prediction for BacteRiAl Proteins (mtx-COBRA). mtx-COBRA uses Meta's protein language model, Evolutionary Scale Modeling, combined with an Extreme Gradient Boosting machine learning model to identify SCL of bacterial proteins based on amino acid sequence. This pipeline is trained on a curated dataset that combines data from UniProt and the publicly available ePSORTdb dataset. RESULTS: Using benchmarking analyses, nested 5-fold cross-validation, and leave-one-pathogen-out methods, followed by testing on the held-out dataset, we show that our pipeline predicts the SCL of bacterial proteins more accurately than PSORTb. CONCLUSIONS: mtx-COBRA provides an accessible pipeline that can more efficiently classify bacterial proteins with currently "Unknown" SCLs than existing bioinformatic and experimental methods.

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

The NLRP3 Inflammasome Is Required for Protection Against Pseudomonas Keratitis.

Purpose: The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of Pseudomonas aeruginosa (PA) infection in mouse corneas. Methods: Corneas of wild type and NLRP3-/- mice were infected with PA. The severity of bacterial keratitis was graded on days 1 and 3 post-infection by slit lamp, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) immunoblotting analysis of cleaved caspase-1 and IL-1ß, and (iv) IL-1ß quantification by ELISA. In parallel experiments, severity of keratitis was examined in the wild-type mice receiving a subconjunctival injection of a highly selective NLRP3 inhibitor immediately prior to infection. Results: Compared to wild type mice, NLRP3-/- mice exhibited more severe infection, as indicated by an increase in opacity score and an increase in bacterial load. The hallmark of inflammasome assembly is the activation of proinflammatory caspase-1 and IL-1ß by cleavage of their precursors, pro-caspase-1 and pro-IL-1ß, respectively. Accordingly, increased severity of infection in the NLRP3-/- mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1ß and reduced IL-1ß+ neutrophil infiltration in infected corneas. Likewise, corneas of mice receiving subconjunctival injections of NLRP3 inhibitor exhibited increased bacterial load, and reduced IL-1ß expression. Conclusions: Activation of NLRP3 pathway is required for the clearance of PA infection in mouse corneas.

Prenylcysteine oxidase 1 like protein is required for neutrophil bactericidal activities.

The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and infection-induced changes in neutrophils. We focus on investigating the Prenylcysteine oxidase 1 like (Pcyox1l) protein function. Murine and human Pcyox1l proteins share ninety four percent aminoacid homology revealing significant evolutionary conservation and implicating Pcyox1l in mediating important biological functions. Here we show that the loss of Pcyox1l protein results in significant reductions in the mevalonate pathway impacting autophagy and cellular viability under homeostatic conditions. Concurrently, Pcyox1l CRISPRed-out neutrophils exhibit deficient bactericidal properties. Pcyox1l knock-out mice demonstrate significant susceptibility to infection with the gram-negative pathogen Psuedomonas aeruginosa exemplified through increased neutrophil infiltrates, hemorrhaging, and reduced bactericidal functionality. Cumulatively, we ascribe a function to Pcyox1l protein in modulation of the prenylation pathway and suggest connections beween metabolic responses and neutrophil functionality.

Inhibition of macrophage migration inhibitory factor ameliorates ocular Pseudomonas aeruginosa-induced keratitis.

Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.

Caveolin-1 modifies the immunity to Pseudomonas aeruginosa.

The inflammatory response to Pseudomonas aeruginosa is not properly regulated in the lungs of patients with cystic fibrosis (CF). In the lung epithelium of individuals with wild-type CF transmembrane conductance regulator, lipid rafts containing CF transmembrane conductance regulator are rapidly formed in response to P. aeruginosa infection, and this response is closely linked to resistance to infection and disease. We found these rafts also contained high levels of caveolin-1 and thus examined the sensitivity of cav1 knockout (KO) mice to P. aeruginosa challenge in both acute and chronic P. aeruginosa infection models. We found that cav1 KO mice had increased sensitivity to P. aeruginosa infection, as represented by an increased mortality rate, elevated bacterial burdens recovered from lungs and spleens, and elevated inflammatory responses. These findings correlated with the decreased ability of cav1-deficient neutrophils to phagocytose P. aeruginosa. In addition, P. aeruginosa colonized cav1 KO mice much better compared with the wild-type controls in a model of chronic infection, indicting an important contribution of Cav-1 to innate host immunity to P. aeruginosa infection in the setting of both acute pneumonia and chronic infection typical of CF.

Analysis of acquisition of Pseudomonas aeruginosa gastrointestinal mucosal colonization and horizontal transmission in a murine model.

BACKGROUND: Laboratory systems to study bacterial transmission and mucosal colonization leading to infection have not been utilized. METHODS: We determined whether transmission of various strains of Pseudomonas aeruginosa among individual mice could occur and investigated the properties of such strains in establishing gastrointestinal (GI) mucosal colonization as well as in disseminating systemically after induction of neutropenia. RESULTS: P. aeruginosa isolates associated with epidemic spread among patients with cystic fibrosis (CF) readily established GI colonization at higher levels than did strains associated with acute infections in patients without CF, and they outcompeted these strains. Colonization was associated with resistance to bile salts. However, epidemic CF isolates did not disseminate after induction of neutropenia and did not induce as much mucosal pathology as did strains that were capable of disseminating. CONCLUSION: Murine models can be used to study P. aeruginosa transmission and early colonization, and the properties of these strains associated with their known clinical behaviors are mimicked in this setting.

Conjunctival Commensal Isolation and Identification in Mice.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an "in-lab" made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment.

Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Murine model of ocular infection Basic Protocol 2: Murine model sample collection Basic Protocol 3: Protein extraction from eye wash Basic Protocol 4: Protein extraction from corneal tissue Basic Protocol 5: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples.