Biology subjects

Schator, D.

Publications and source records attributed to Schator, D..

5 recordsLinked to original sources

bioRxiv2025

Cross-membrane cooperation among bacteria can facilitate intracellular pathogenesis

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause life- and sight-threating infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that P. aeruginosa inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca2+ influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among P. aeruginosa subpopulations can contribute to disseminating intracellular bacteria in vivo in a mouse infection model. This study provides the basis for future studies to investigate how cooperation of extracellular and intracellular bacteria within the host may contribute to disease progression and persistence.

microbiology↗

bioRxiv2025

The Pseudomonas aeruginosa T3SS can contribute to traversal of an in situ epithelial multilayer independently of the T3SS needle

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a {Delta}pscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a {Delta}exsD mutant constitutively on for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes ({Delta}pscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted ({Delta}pscU-L{Delta}exoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented {Delta}pscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to development of therapeutics targeting the T3SS system. IO_SCPLOWMPORTANCEC_SCPLOWWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations of in vivo/in situ models and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

microbiology↗

bioRxiv2024

Contact Lens Wear Alters Transcriptional Responses to Pseudomonas aeruginosa in Both the Corneal Epithelium and the Bacteria

PurposeHealthy corneas resist colonization by virtually all microbes yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. MethodsMale and female C57BL/6J mice were fitted with a contact lens on one eye for 24 h. After lens removal, corneas were immediately challenged for 4 h with P. aeruginosa. A separate group of naive mice were similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naive controls as was lens wear alone. Total RNA-sequencing determined corneal epithelium and bacterial gene expression. ResultsPrior lens wear profoundly altered the corneal response to P. aeruginosa, including: upregulated pattern-recognition receptors (tlr3, nod1), downregulated lectin pathway of complement activation (masp1), amplified upregulation of tcf7, gpr55, ifi205, wfdc2 (immune defense) and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, gpr55 (immune defense) and downregulated Ca2+-dependent genes necab1, snx31 and npr3. P. aeruginosa exposure to prior lens wearing vs. naive corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226) and antimicrobial resistance (arnB, oprR). ConclusionPrior lens wear impacts corneal epithelium gene expression altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.

microbiology↗

bioRxiv2022

Legionella para-effectors target chromatin and promote bacterial replication

Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a {Delta}lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout ({Delta}lphD{Delta}romA) can restore intracellular replication. Uniquely, we present evidence for "para-effectors", an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens opens new vistas for the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences.

microbiology↗

bioRxiv2021

Zebrafish larvae as a powerful model to dissect protective innate immunity in response to Legionella pneumophila infection

The zebrafish has become a powerful model organism to study host-pathogen interactions. Here, we developed a zebrafish model of Legionella pneumophila infection to dissect innate immune responses. We show that L. pneumophila cause zebrafish larvae death in a dose dependent manner, and that macrophages are the first line of defence, with neutrophils cooperating to clear the infection. When either macrophages or neutrophils are depleted, these "immunocompromised" larvae become lethally sensitive to L. pneumophila similar to what is known for humans that develop pneumonia. Also as observed in human infections, the adaptor signalling molecule Myd88 is not required to control disease in the larvae. Furthermore, proinflammatory cytokine genes il1{beta} and tnf were upregulated during infection, recapitulating key immune responses seen in human infection. Strikingly, we uncovered a previously undescribed infection phenotype in zebrafish larvae, whereby bloodborne, wild type L. pneumophila invade and grow in the larval yolk region, a phenotype not observed with a type IV secretion system deficient mutant that cannot translocate effectors into its host cell. Thus, zebrafish larva represents an innovative L. pneumophila infection model that that on one hand mimics important aspects of the human immune response to L. pneumophila infection and that on the other hand will allow to elucidate the mechanisms by which type IV secretion effectors allow L. pneumophila to cross membranes and to obtain nutrients from nutrient rich environments.

microbiology↗