Biology subjects

Pearlman, E.

Publications and source records attributed to Pearlman, E..

8 recordsLinked to original sources

bioRxiv2026

Spatial transcriptomics identifies the Pseudomonas aeruginosa major outer membrane protein OprF as a critical factor in blinding corneal infections

Pseudomonas aeruginosa is a globally recognized pathogen causing pulmonary, skin, and severe corneal infections (keratitis), with the potential to induce irreversible blindness if untreated. Spatial transcriptomic analysis of P. aeruginosa infected corneas identified elevated expression of the outer membrane proteins OprF and OprL and PA1414, which encodes the small RNA SicX in the corneal stroma compared with corneal epithelium. Comparative spatial transcriptomics analysis of corneas infected with an oprF transposon (TN) mutant showed reduced expression of the type III effector protein ExoT, which was absent in an oprF deficient mutant ({Delta}oprF) and in contrast to PA14, did not inhibit reactive oxygen species (ROS) production by neutrophils. Corneal infection with the {Delta}oprF mutant resulted in reduced corneal virulence and lower CFU compared to the parental PA14 strain. Collectively, our findings demonstrate a coordinated virulence program connecting OprF functionality with the release of ExoT and its ability to block ROS production and survive in infected corneas.

microbiology↗

bioRxiv2026

Phagocytic Clearance of SARS-CoV-2 Nucleocapsid- and RNA-Containing Immune Complexes Drives Inflammatory Cytokine Production and Endothelial Dysfunction

The aberrant inflammation that characterizes severe COVID-19 is incompletely understood. Given the persistence of SARS-Cov-2 RNA and nucleocapsid protein (N) and the presence of anti-N antibody during the course of severe infection, we investigated the role of RNA-containing immune complexes (ICs) in driving inflammation. We found that ICs consisting of SARS-CoV-2 RNA, N, and anti-N IgG1 are internalized by primary human monocytes and stimulate inflammatory cytokines and chemokines in a manner dependent on Fc{gamma} receptors and partially dependent on toll-like receptor-8. In addition, the inflammatory response induced in monocytes by RNA-containing ICs caused endothelial dysfunction in vascularized micro-organs. Using nasopharyngeal samples from SARS-CoV-2-infected individuals, SARS-CoV-2 RNA and N were captured by anti-N monoclonal antibody in the absence of lysing reagents, indicating that SARS-CoV-2 RNA and N complexes are present outside of virions and cells. Finally, we found that during an early wave of COVID-19, higher anti-N IgG:IgM ratios were associated with more severe clinical outcomes, consistent with a role for inflammatory, IgG-mediated phagocytic clearance of nucleic acid-containing ICs in SARS-CoV-2 pathogenesis, perhaps mitigated by non-inflammatory, IgM-mediated clearance. We conclude that RNA-containing ICs may play a role in the pathogenesis of severe COVID-19. Since all pathogenic viruses encode nucleic acid-binding proteins, such as N, and these proteins often elicit an antibody response, inflammatory clearance of nucleic acid-containing ICs may also contribute to disease severity in other viral infections.

immunology↗

bioRxiv2026

Pseudomonas aeruginosa Exotoxin A is not essential for corneal disease severity or bacterial survival

Pseudomonas aeruginosa produces multiple toxins and exoenzymes that contribute to its survival and ability to cause disease. As prior studies reported an important role for type III-secretion in the severity of corneal disease in P. aeruginosa keratitis, we examined if there is a requirement for the type II-secreted cytotoxin Exotoxin A (ToxA) in bacterial persistence and disease severity in infected murine corneas. Using independently generated {Delta}toxA mutants and complemented strains, we found that ToxA is expressed in vivo, but ToxA deletion did not significantly affect bacterial replication, neutrophil recruitment or disease severity. These findings contrast with an earlier study identifying a critical role for ToxA.

microbiology↗

bioRxiv2024

A biofilm-tropic Pseudomonas aeruginosa bacteriophage uses the exopolysaccharide Psl as receptor

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

microbiology↗

bioRxiv2024

An essential role for the Hv1 voltage-gated proton channel in Pseudomonas aeruginosa corneal infection

Assembly of NADPH oxidase 2 (NOX2) proteins in neutrophils plays an essential role in controlling microbial infections by producing high levels of reactive oxygen species (ROS). We reported that neutrophils and NOX2 are required to control P. aeruginosa in a clinically relevant murine model of blinding corneal infection. Given the published role for the voltage-gated proton channel Hv1 in sustaining NOX2 production, we examined the role of Hv1 in P. aeruginosa keratitis. Hvcn1-/- mice exhibited an impaired ability to kill bacteria that was associated with reduced neutrophil recruitment to infected corneas. Unlike earlier reports, we found that Hvcn1-/- neutrophils produce more rather than less ROS compared with control neutrophils infected with P. aeruginosa or stimulated with PMA or zymosan. Collectively, we demonstrate that Hv1 has an important role in control of bacterial growth by neutrophils in bacterial infection beyond the regulation of ROS production.

microbiology↗

bioRxiv2024

Spatial transcriptomics identifies novel P. aeruginosa virulence factors

To holistically unravel the complexity of pathogen-host interactions within infected tissues we leverage a dual spatial transcriptomic approach that, for the first time, simultaneously captures the expression of Pseudomonas aeruginosa genes alongside the entire host transcriptome in a model of ocular infection. This innovative method reveals differential pathogen and host-specific gene expression patterns across specific anatomical regions generating a unified transcriptional map of infection. By integrating these data, we developed a predictive ridge regression model trained on images from infected tissues. The model achieved an R{superscript 2} score of 0.923 in predicting bacterial burden distributions by using host features thereby predicting novel biomarkers associated with disease severity. Our analysis revealed a complex interplay between P. aeruginosa nutritional requirements and protective host responses and identified novel interactions between bacterial metabolite transport proteins and host autophagy. Among an array of iron acquisition gene transcripts that showed significant enrichment at the host-pathogen interface, we discovered a novel virulence mediator PA2590. This study highlights the power of spatial transcriptomics, particularly in combining bacterial and host transcriptomes, to uncover novel host-pathogen interactions, advance our understanding of bacterial virulence mechanisms, and point to druggable molecules.

microbiology↗

bioRxiv2022

Neutrophil IL-1β secretion induced by ExoS expressing Pseudomonas aeruginosa is dependent on NLRP3 and Gasdermin D

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1{beta} secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1{beta} secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ({Delta}exoST) stimulated NLRC4 for IL-1{beta} secretion. While IL-1{beta} release from{Delta} exoST infected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model of P. aeruginosa corneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

microbiology↗

bioRxiv2020

Control of ss-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence

Candida albicans is a major cause of invasive candidiasis, which has a high mortality rate. The hyphal form of C. albicans is virulent and activates the host innate immune response, while the yeast form is hypovirulent and less immunogenic. The innate immune response is critical for host defense, but overactivation can cause tissue damage and sepsis. The innate immune response can be triggered when the C-type lectin receptor Dectin-1 recognizes {beta}-glucans, which is protected by the outer mannan layer of the cell wall on C. albicans. Here, we demonstrate that there is low level of Dectin-1 binding at the septum of yeast cells, but high level of Dectin-1 binding over the entire surface of hyphae. We find that {beta}-glucan masking in yeast is controlled by two highly expressed yeast proteins, the endo-1,3-{beta}-glucanase Eng1 and the Yeast Wall Protein Ywp1. An eng1 deletion mutant shows enhanced Dectin-1 binding at the septa, while an eng1 ywp1 double mutant, but not an ywp1 single mutant, shows strong overall Dectin-1 binding. Thus, Eng1-mediated {beta}-glucan trimming and Ywp1-mediated {beta}-glucan masking are two parallel mechanisms utilized by C. albicans yeast to minimize recognition by Dectin-1. In the model of disseminated candidiasis, mice infected with the eng1 deletion mutant showed delayed mortality with an increased renal immune response in males compared to mice infected with the wild-type strain, but earlier mortality with a higher renal immune response in females. Using the eng1 mutant that is specifically defective in {beta}-glucan masking in yeast, this study demonstrates that the level of {beta}-glucan exposure is important for modulating the balance between immune protection and immunopathogenesis. Abstract ImportanceCandida albicans is a major opportunistic fungal pathogen of humans. Systemic Candidiasis has high mortality rates. C. albicans is also a constituent of the human microbiome and found in gastrointestinal and genitourinary tracts of most healthy individuals. C. albicans is able to switch reversibly between yeast and hyphae in response to environmental cues. The hyphal form is virulent, while the yeast form is hypovirulent and less immunogenic. This study demonstrates that {beta}-glucan exposure in yeast is protected by two highly expressed yeast proteins, the endo-1,3-{beta}-glucanase Eng1 and the Yeast Wall Protein Ywp1. Eng1-mediated {beta}-glucan trimming and Ywp1-mediated {beta}-glucan masking are two parallel mechanisms utilized by C. albicans yeast to minimize recognition by the host C-type lectin receptor Dectin-1. The eng1 mutant triggers a higher immune response and leads to earlier mortality compared to the wild-type strain. Thus, {beta}-glucan masking in yeast keeps yeast cells less immunogenic and hypovirulent.

microbiology↗