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Purpose: To explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis. Methods: Bacillus endophthalmitis was induced in mice. Innate immune pathway activation was interfered by injecting S layer protein-deficient (∆slpA) B. thuringiensis or by treating wild-type (WT)–infected mice with a TLR2/4 inhibitor (WT+OxPAPC). At 10 hours postinfection, eyes were harvested and RNA was purified. A NanoString murine inflammation panel was used to compare gene expression in WT-infected, WT+OxPAPC, ∆slpA-infected, and uninfected eyes. Results: In WT-infected eyes, 56% of genes were significantly upregulated compared to uninfected controls. Compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ∆slpA-infected eyes, respectively. Expression of 61 genes that were upregulated in WT-infected eyes was decreased in WT+OxPAPC and ∆slpA-infected eyes. Innate interference resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate pathway genes (TLRs 2, 4, 6, and 8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2). Innate interference also reduced the expression of several inflammatory cytokines (CSF2, CSF3, IL-6, IL-1β, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β) and chemokines (CCL2, CCL3, and CXCLs 1, 2, 3, 5, 9, and 10). All of the aforementioned genes were significantly upregulated in WT-infected eyes. Conclusions: These results suggest that interfering with innate activation significantly reduced the intraocular inflammatory response in Bacillus endophthalmitis. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.
Huzzatul Mursalin; Phillip S. Coburn; Frederick C. Miller; Erin T. Livingston; Roger Astley; Michelle C. Callegan. Innate Immune Interference Attenuates Inflammation InBacillusEndophthalmitis. Investigative Opthalmology & Visual Science 2020, 61, 17 -17.
AMA StyleHuzzatul Mursalin, Phillip S. Coburn, Frederick C. Miller, Erin T. Livingston, Roger Astley, Michelle C. Callegan. Innate Immune Interference Attenuates Inflammation InBacillusEndophthalmitis. Investigative Opthalmology & Visual Science. 2020; 61 (13):17-17.
Chicago/Turabian StyleHuzzatul Mursalin; Phillip S. Coburn; Frederick C. Miller; Erin T. Livingston; Roger Astley; Michelle C. Callegan. 2020. "Innate Immune Interference Attenuates Inflammation InBacillusEndophthalmitis." Investigative Opthalmology & Visual Science 61, no. 13: 17-17.
Bacterial endophthalmitis is a devastating infection that can cause blindness following the introduction of organisms into the posterior segment of the eye. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Often, these eyes have to be enucleated. Bacillus produces many virulence factors in the eye that may contribute to retinal damage and robust inflammation. This study analyzed Bacillus immune inhibitor A (InhA) metalloproteases, which digest extracellular matrix, tight junction proteins, and antimicrobial proteins. We hypothesized that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared between B. thuringiensis strains. WT and InhA mutants were similarly cytotoxic to retinal cells. Mutant ΔinhA1 and ΔinhA2 entered log phase growth earlier than WT. Proteolysis of the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of B. thuringiensis WT or InhA mutants. Intraocular Bacillus and retinal function loss were quantified. Intraocular myeloperoxidase concentrations were quantified and histology was analyzed. Eyes infected with ΔinhA1 or ΔinhA2 strains contained greater numbers of bacteria than eyes infected with WT throughout the course of infection. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with WT strain. Eyes infected with ΔinhA1-3 cleared the infection, with less retinal function loss and inflammation compared to eyes infected with the WT strain. RT-PCR results suggested that single InhA mutant results may be explained by compensatory expression of the other InhAs in these mutants. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.
Erin T Livingston; Huzzatul Mursalin; Phillip S Coburn; Roger Astley; Frederick C Miller; Omar Amayem; Didier Lereclus; Michelle Callegan. Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis. 2020, 1 .
AMA StyleErin T Livingston, Huzzatul Mursalin, Phillip S Coburn, Roger Astley, Frederick C Miller, Omar Amayem, Didier Lereclus, Michelle Callegan. Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis. . 2020; ():1.
Chicago/Turabian StyleErin T Livingston; Huzzatul Mursalin; Phillip S Coburn; Roger Astley; Frederick C Miller; Omar Amayem; Didier Lereclus; Michelle Callegan. 2020. "Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis." , no. : 1.
Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To begin to better understand the role of other B. cereus virulence genes in endophthalmitis, expression levels of a subset of factors was measured at the midpoint of disease progression in a murine model of experimental endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY, gntR, and nprR were also expressed in vivo. However, at this timepoint, plcR/papR was not detectable, we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla, fliF, and motB, and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expression gene in vivo, suggesting that this gene is criticial for intraocular survival, potentially through inhibition of neutrophil activity. Expression of the surface layer protein gene, slpA, an activator of Toll-like receptors (TLR)-2 and -4, and a potent contributor to intraocular inflammation and disease severvity, was also detected during infection, albeit at low levels. In summary, genes expressed in a mouse model of Bacillus endophthalmitis might prove to play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed attenuate the severity of this often blinding infection.
Phillip S Coburn; Frederick C Miller; Morgan A Enty; Craig Land; Austin L LaGrow; Huzzatul Mursalin; Michelle C Callegan. The Bacillus Virulome in Endophthalmitis. 2020, 1 .
AMA StylePhillip S Coburn, Frederick C Miller, Morgan A Enty, Craig Land, Austin L LaGrow, Huzzatul Mursalin, Michelle C Callegan. The Bacillus Virulome in Endophthalmitis. . 2020; ():1.
Chicago/Turabian StylePhillip S Coburn; Frederick C Miller; Morgan A Enty; Craig Land; Austin L LaGrow; Huzzatul Mursalin; Michelle C Callegan. 2020. "The Bacillus Virulome in Endophthalmitis." , no. : 1.
PURPOSE Bacillusendophthalmitis is a sight-threatening bacterial infection that sometimes requires enucleation. Inflammation in this disease is driven by activation of innate Toll-like receptor (TLR) pathways. Here, we explored the consequences of innate immune interference on intraocular inflammatory responses duringBacillusendophthalmitis. METHODS Endophthalmitis was induced in mice by injecting 100 CFUBacillus thuringiensisin to the mid-vitreous. We interfered with activation of the TLR2 and TLR4 pathways by 1) injecting a group of mice with S layer protein-deficient (ΔslpA)B. thuringiensisor 2) injecting a group of wild type (WT)-infected mice with a TLR2/4 inhibitor, oxidized phospholipid (OxPAPC). At 10 hours postinfection, infected eyes were removed and total RNA was purified. mRNA expression was then analyzed by NanoString using a murine inflammation panel. We compared findings with expression data from eyes infected with eyes injected with WTB. thuringiensis, eyes injected with OxPAPC alone, and uninfected eyes. RESULTS Interference of TLR2 and TLR4 pathways resulted in differential expression of mouse inflammatory genes compared to expression in WT-infected eyes. In WT-infected eyes, 56% of genes were significantly upregulated compared to that of uninfected controls. However, compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ΔslpA-infected eyes, respectively. The expression of 61 genes which were significantly upregulated in WT-infected eyes was decreased in WT+OxPAPC or ΔslpA-infected eyes. Interference with activation of the TLR2 and TLR4 pathways resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate genes such as TLR2, TLR4, TLR6, TLR8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2. Interference with activation of the TLR2 and TLR4 pathways also reduced the expression of several inflammatory cytokines such as CSF3, IL-6, IL-1β, CSF2, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β and chemokines CCL2, CCl3, CXCL1, CXCL2, CXCL3, CXCL5, CXCL9, and CXCL10. All of the aforementioned genes were significantly upregulated in WT-infected eyes. CONCLUSIONS These results suggest that interfering with the activation of innate immune pathways duringBacillusendophthalmitis significantly reduced the intraocular inflammatory response. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.
Huzzatul Mursalin; Phillip S. Coburn; Frederick C. Miller; Erin Livingston; Roger Astley; Michelle C. Callegan. INNATE IMMUNE INTERFERENCE ATTENUATES INFLAMMATION INBACILLUSENDOPHTHALMITIS. bioRxiv 2020, 1 .
AMA StyleHuzzatul Mursalin, Phillip S. Coburn, Frederick C. Miller, Erin Livingston, Roger Astley, Michelle C. Callegan. INNATE IMMUNE INTERFERENCE ATTENUATES INFLAMMATION INBACILLUSENDOPHTHALMITIS. bioRxiv. 2020; ():1.
Chicago/Turabian StyleHuzzatul Mursalin; Phillip S. Coburn; Frederick C. Miller; Erin Livingston; Roger Astley; Michelle C. Callegan. 2020. "INNATE IMMUNE INTERFERENCE ATTENUATES INFLAMMATION INBACILLUSENDOPHTHALMITIS." bioRxiv , no. : 1.
Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence.
Phillip S. Coburn; Frederick C. Miller; Morgan A. Enty; Craig Land; Austin L. LaGrow; Huzzatul Mursalin; Michelle C. Callegan. Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment. Microorganisms 2020, 8, 607 .
AMA StylePhillip S. Coburn, Frederick C. Miller, Morgan A. Enty, Craig Land, Austin L. LaGrow, Huzzatul Mursalin, Michelle C. Callegan. Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment. Microorganisms. 2020; 8 (4):607.
Chicago/Turabian StylePhillip S. Coburn; Frederick C. Miller; Morgan A. Enty; Craig Land; Austin L. LaGrow; Huzzatul Mursalin; Michelle C. Callegan. 2020. "Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment." Microorganisms 8, no. 4: 607.
Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we had reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence.Impact statementB. cereus is the causative agent of gastrointestinal infections, but can also cause a serious infection of the eye that often results in blindness or enucleation. Current therapeutic measures often fail to mitigate these poor outcomes. This necessitates the development of new treatment modalities based on new targets. To begin to better define those B. cereus factors with roles in intraocular infection, we analyzed the expression of genes related to gastrointestinal infections, as well as those with both known and hypothesized roles in intraocular infections, after growth in an ex vivo vitreous. Potentially targetable candidate genes were demonstrated to be expressed in vitreous, which suggests that these genes might contribute to the unique virulence of B. cereus endophthalmitis. Importantly, our results lay the groundwork for assessing the expression of these genes in vivo and defining the virulome of B. cereus in intraocular infections.
Phillip S. Coburn; Frederick C. Miller; Morgan A. Enty; Craig Land; Austin L. LaGrow; Huzzatul Mursalin; Michelle C. Callegan. Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment. 2020, 1 .
AMA StylePhillip S. Coburn, Frederick C. Miller, Morgan A. Enty, Craig Land, Austin L. LaGrow, Huzzatul Mursalin, Michelle C. Callegan. Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment. . 2020; ():1.
Chicago/Turabian StylePhillip S. Coburn; Frederick C. Miller; Morgan A. Enty; Craig Land; Austin L. LaGrow; Huzzatul Mursalin; Michelle C. Callegan. 2020. "Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment." , no. : 1.
Some tissues of the eye are susceptible to damage due to their exposure to the outside environment and inability to regenerate. Immune privilege, although beneficial to the eye in terms of homeostasis and protection, can be harmful when breached or when an aberrant response occurs in the face of challenge. In this review, we highlight the role of the PMN (polymorphonuclear leukocyte) in different bacterial ocular infections that invade the immune privileged eye at the anterior and posterior segments: keratitis, conjunctivitis, uveitis, and endophthalmitis. Interestingly, the PMN response from the host seems to be necessary for pathogen clearance in ocular disease, but the inflammatory response can also be detrimental to vision retention. This “Pyrrhic Victory” scenario is explored in each type of ocular infection, with details on PMN recruitment and response at the site of ocular infection. In addition, we emphasize the differences in PMN responses between each ocular disease and its most common corresponding bacterial pathogen. The in vitro and animal models used to identify PMN responses, such as recruitment, phagocytosis, degranulation, and NETosis, are also outlined in each ocular infection. This detailed study of the ocular acute immune response to infection could provide novel therapeutic strategies for blinding diseases, provide more general information on ocular PMN responses, and reveal areas of bacterial ocular infection research that lack PMN response studies.
Erin T. Livingston; Huzzatul Mursalin; Michelle C. Callegan. A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections. Microorganisms 2019, 7, 537 .
AMA StyleErin T. Livingston, Huzzatul Mursalin, Michelle C. Callegan. A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections. Microorganisms. 2019; 7 (11):537.
Chicago/Turabian StyleErin T. Livingston; Huzzatul Mursalin; Michelle C. Callegan. 2019. "A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections." Microorganisms 7, no. 11: 537.
Staphylococcus aureus (S. aureus) is a common pathogen of the eye, capable of infecting external tissues such as the tear duct, conjunctiva, and the cornea, as well the inner and more delicate anterior and posterior chambers. S. aureus produces numerous toxins and enzymes capable of causing profound damage to tissues and organs, as well as modulating the immune response to these infections. Unfortunately, in the context of ocular infections, this can mean blindness for the patient. The role of α-toxin in corneal infection (keratitis) and infection of the interior of the eye (endophthalmitis) has been well established by comparing virulence in animal models and α-toxin-deficient isogenic mutants with their wild-type parental strains. The importance of other toxins, such as β-toxin, γ-toxin, and Panton–Valentine leukocidin (PVL), have been analyzed to a lesser degree and their roles in eye infections are less clear. Other toxins such as the phenol-soluble modulins have yet to be examined in any animal models for their contributions to virulence in eye infections. This review discusses the state of current knowledge of the roles of S. aureus toxins in eye infections and the controversies existing as a result of the use of different infection models. The strengths and limitations of these ocular infection models are discussed, as well as the need for physiological relevance in the study of staphylococcal toxins in these models.
Roger Astley; Frederick C. Miller; Huzzatul Mursalin; Phillip S. Coburn; Michelle C. Callegan. An Eye on Staphylococcus aureus Toxins: Roles in Ocular Damage and Inflammation. Toxins 2019, 11, 356 .
AMA StyleRoger Astley, Frederick C. Miller, Huzzatul Mursalin, Phillip S. Coburn, Michelle C. Callegan. An Eye on Staphylococcus aureus Toxins: Roles in Ocular Damage and Inflammation. Toxins. 2019; 11 (6):356.
Chicago/Turabian StyleRoger Astley; Frederick C. Miller; Huzzatul Mursalin; Phillip S. Coburn; Michelle C. Callegan. 2019. "An Eye on Staphylococcus aureus Toxins: Roles in Ocular Damage and Inflammation." Toxins 11, no. 6: 356.