The Transcriptional Regulator SpxA1 Influences the Morphology and Virulence of
            <i>Listeria monocytogenes</i>

Cesinger, Monica R.; Daramola, Oluwasegun I.; Kwiatkowski, Lucy M.; Reniere, Michelle L.

doi:10.1128/iai.00211-22

“…Interestingly, uptake of Lm was unchanged in Tlr2 −/− BMDMs compared to WT BMDMs (S4B Fig). While we did not directly test Tlr5 −/− macrophages, studies from our lab have found that Lm lacking the TLR5 ligand flaA are taken up by macrophages with equal efficiency as wildtype Lm (40). To account for the possibility of redundancy between TLR2 and TLR5, we infected wildtype and Tlr2 −/− BMDMs with Δ flaA and found that uptake remained unchanged in the absence of TLR2 and TLR5 recognition (S4C Fig).…”

Section: Resultsmentioning

confidence: 93%

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

Glover

¹

,

Schwardt

²

,

Sanchez

³

et al. 2022

Preprint

0

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Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised individuals. Macrophages play a dual role during Lm infection by both promoting dissemination of Lm from the gastrointestinal tract and limiting bacterial growth upon immune activation. Despite the relevance of macrophages to Lm infection, the mechanisms underlying phagocytosis of Lm by macrophages are not well understood. To identify host factors important for Lm infection of macrophages, we performed an unbiased CRISPR/Cas9 screen which revealed pathways that are specific to phagocytosis of Lm and those that are required for internalization of bacteria generally. Specifically, we discovered the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, we found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, we show that PTEN-dependent phagocytosis is important for host protection during oral Lm infection. Overall, this study provides a comprehensive identification of macrophage factors involved in regulating Lm uptake and characterizes the function of one factor, PTEN, during Lm infection in vitro and in vivo. Importantly, these results demonstrate a role for opsonin-independent phagocytosis in Lm pathogenesis and suggest that macrophages play a primarily protective role during foodborne listeriosis.

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“…Following elution, peptides were completely dried under vacuum and resuspended in 0.1% (v/v) formic acid to a final concentration of ∼0.5 µg/mL. Samples were transferred into autosampler vials and subjected to mass spectrometry analysis using an Orbitrap Eclipse mass spectrometer (Thermo Scientific) as described (101). Raw spectral data were processed in MaxQuant using the Homo sapiens reference protein library (UniProt) and relative protein abundances were determined using the label-free quantification (LFQ) intensity method as described (101).…”

Section: Methodsmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted March 7, 2023. ; https://doi.org/10.1101/2023.03.07.531620 doi: bioRxiv preprint 22 described (101). Raw spectral data were processed in MaxQuant using the Homo sapiens reference protein library (UniProt) and relative protein abundances were determined using the label-free quantification (LFQ) intensity method as described (101).…”

Section: Whole-cell Proteomicsmentioning

confidence: 99%

Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

Zaver

¹

,

Sarkar

²

,

Egolf

³

et al. 2023

Preprint

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Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than two decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2 knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomic analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAP kinase signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings with lesions showing keratin deficiency, cadherin mis-localization, and ERK hyper-phosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multi-omic analysis with human organotypic epidermis as a pre-clinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.

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“…To test this hypothesis, we measured uptake of Lm and PI3K activation in Tlr2 -/macrophages. For these studies, BMDMs from Tlr2 -/mice were isolated and absence of TLR2 was confirmed by immunoblot analysis (S5A Fig) . Interestingly, uptake of Lm was unchanged in Tlr2 -/-BMDMs compared to WT BMDMs (S5B Fig) . While we did not directly test Tlr5 -/macrophages, studies from our lab have found that Lm lacking the TLR5 ligand flaA are taken up by macrophages with equal efficiency as wildtype Lm [40]. To account for the possibility of redundancy between TLR2 and TLR5, we infected wildtype and Tlr2 -/-BMDMs with ΔflaA and found that uptake remained unchanged in the absence of TLR2 and TLR5 recognition (S5C Fig) . These results indicate that phagocytosis of Lm is TLR-independent.…”

Section: Class 1a Pi3k Activation Is Required For Lm Phagocytosis By ...mentioning

confidence: 99%

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction of Listeria monocytogenes infection

Glover

¹

,

Schwardt

²

,

Sanchez

³

et al. 2023

Self Cite

View full text Add to dashboard Cite

Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised individuals. Macrophages play a dual role during Lm infection by both promoting dissemination of Lm from the gastrointestinal tract and limiting bacterial growth upon immune activation. Despite the relevance of macrophages to Lm infection, the mechanisms underlying phagocytosis of Lm by macrophages are not well understood. To identify host factors important for Lm infection of macrophages, we performed an unbiased CRISPR/Cas9 screen which revealed pathways that are specific to phagocytosis of Lm and those that are required for internalization of bacteria generally. Specifically, we discovered the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, we found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, we show that PTEN-dependent phagocytosis is important for host protection during oral Lm infection. Overall, this study provides a comprehensive identification of macrophage factors involved in regulating Lm uptake and characterizes the function of one factor, PTEN, during Lm infection in vitro and in vivo. Importantly, these results demonstrate a role for opsonin-independent phagocytosis in Lm pathogenesis and suggest that macrophages play a primarily protective role during foodborne listeriosis.

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The Transcriptional Regulator SpxA1 Influences the Morphology and Virulence of Listeria monocytogenes

Cited by 5 publications

References 52 publications

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction ofListeria monocytogenesinfection

Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction of Listeria monocytogenes infection

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