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Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

ABSTRACT: Pathogenic bacteria have evolved diverse mechanisms to counteract cell-autonomous immunity, which otherwise guards both immune and non-immune cells from the onset of an infection^1,2. The versatile immunity protein Ring finger protein 213 (RNF213)^3-6 mediates the non-canonical ester-linked ubiquitylation of lipopolysaccharide (LPS), marking bacteria that sporadically enter the cytosol for destruction by antibacterial autophagy⁴. However, whether cytosol-adapted pathogens are ubiquitylated on their LPS and whether they escape RNF213-mediated immunity, remains unknown. Here we show that Burkholderia deubiquitylase (DUB), TssM^7-9, is a potent esterase that directly reverses the ubiquitylation of LPS. Without TssM, cytosolic Burkholderia became coated in polyubiquitin and autophagy receptors in an RNF213-dependent fashion. Whereas the expression of TssM was sufficient to enable the replication of the non-cytosol adapted pathogen Salmonella, we demonstrate that Burkholderia has evolved a multi-layered defence system to proliferate in the host cell cytosol, including a block in antibacterial autophagy^10-12. Structural analysis provided insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from isopeptidase function. TssM homologs conserved in another Gram-negative pathogen also reversed non-canonical LPS ubiquitylation, establishing esterase activity as a bacterial virulence mechanism to subvert host cell-autonomous immunity.

SUBMITTER: Szczesna M

PROVIDER: S-EPMC10371091 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

Szczesna Magdalena M Huang Yizhou Y Lacoursiere Rachel E RE Bonini Francesca F Pol Vito V Koc Fulya F Ward Beatrice B Geurink Paul P PP Pruneda Jonathan N JN Thurston Teresa L M TLM

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Pathogenic bacteria have evolved diverse mechanisms to counteract cell-autonomous immunity, which otherwise guards both immune and non-immune cells from the onset of an infection1,2. The versatile immunity protein Ring finger protein 213 (RNF213)3-6 mediates the non-canonical ester-linked ubiquitylation of lipopolysaccharide (LPS), marking bacteria that sporadically enter the cytosol for destruction by antibacterial autophagy4. However, whether cytosol-adapted pa ...[more]

PMID: 37503018

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Project description:Depending on the specific viral pathogen, commensal bacteria can promote or reduce the severity of viral infection and disease progression in their hosts. Influenza A virus (IAV) has a broad host range, comprises many subtypes, and utilizes different routes of transmission, including the fecal-oral route in wild birds. It has been previously demonstrated that commensal bacteria can interact with the host's immune system to protect against IAV pathogenesis. However, it is unclear whether bacteria and their products may be interacting directly with IAV to impact virion stability. Herein we show that gastrointestinal (GI) tract bacterial isolates in an in vitro system significantly reduce the stability of IAV. Moreover, bacterial lipopolysaccharide (LPS), found on the exterior surfaces of bacteria, was sufficient to significantly decrease the stability of both human and avian viral strains in a temperature-dependent manner, including at the relevant temperatures of their respective hosts and the external aquatic habitat. The subtype and host origin of the viruses were shown to affect the extent to which IAV was susceptible to LPS. Furthermore, using a receptor binding assay and transmission electron microscopy, we observed that LPS binds to and alters the morphology of influenza virions, suggesting that direct interaction with the viral surface contributes to the observed antiviral effect of LPS on influenza. IMPORTANCE Influenza A virus (IAV), transmitted primarily via the fecal-oral route in wild birds, encounters high concentrations of bacteria and their products. Understanding the extent to which bacteria affect the infectivity of IAV will lead to a broader understanding of viral ecology in reservoir hosts and may lead to insights for the development of therapeutics in respiratory infection. Herein we show that bacteria and lipopolysaccharide (LPS) interact with and destabilize influenza virions. Moreover, we show that LPS reduces the long-term persistence and freeze-thaw stability of IAV, which is important information for modeling the movement and emergence of novel strains from animal hosts. Our results, demonstrating that the subtype and host origin of a virus also influence its susceptibility to LPS, raise key questions about the fitness of viruses in reservoir hosts, their potential to transmit to humans, and the importance of bacterial-viral interactions in viral ecology.

Project description:BackgroundLignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration.ResultsTen CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass.ConclusionsBacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin-carbohydrate disassembly.

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Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

Publications

Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

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