Dining in: intracellular bacterial pathogen interplay with autophagy

Intracellular bacterial pathogens have evolved many ways to manipulate host cells for successful infection. Many of these pathogens use specialized secretion systems to inject bacterial proteins into the host cytosol that manipulate cellular processes to favor infection. Autophagy is a eukaryotic cellular remodeling process with a critical role in many diseases, including bacterial clearance. A growing field of research highlights mechanisms used by intracellular bacteria to manipulate autophagy as a pro-survival strategy. This review focuses on a select group of bacterial pathogens with diverse intracellular lifestyles that exploit autophagy-derived nutrients and membrane for survival. This group of pathogens uses secretion systems and specific effectors to subvert distinct components of autophagy. By understanding how intracellular pathogens manipulate autophagy, we gain insight not only into bacterial pathogenesis but also host cell signaling and autophagolysosome maturation

Evaluation of IL-1 Blockade as an Adjunct to Linezolid Therapy for Tuberculosis in Mice and Macaques

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during M. tuberculosis infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area

Evaluation of IL-1 blockade as a host-directed therapy for tuberculosis in mice and macaques [preprint]

In 2017, there were over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB), emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for antibiotic-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, a pathway important for early immunity during M. tuberculosis infection that later contributes to pathology. We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce BM toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, mice and cynomolgus macaques. Antagonizing IL-1 in chronically-infected mice reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of BM suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 1 month, the majority of granulomas were sterilized with reduced inflammation (assessed by PET/CT) in animals treated with both LZD and IL-1 receptor antagonist (IL-1Rn). However, overall lung inflammation was significantly reduced in macaques treated with both IL-1Rn and LZD, compared to LZD alone. Importantly, IL-1Rn administration did not noticeably impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection

Dining in: intracellular bacterial pathogen interplay with autophagy

Intracellular bacterial pathogens have evolved many ways to manipulate host cells for successful infection. Many of these pathogens use specialized secretion systems to inject bacterial proteins into the host cytosol that manipulate cellular processes to favor infection. Autophagy is a eukaryotic cellular remodeling process with a critical role in many diseases, including bacterial clearance. A growing field of research highlights mechanisms used by intracellular bacteria to manipulate autophagy as a pro-survival strategy. This review focuses on a select group of bacterial pathogens with diverse intracellular lifestyles that exploit autophagy-derived nutrients and membrane for survival. This group of pathogens uses secretion systems and specific effectors to subvert distinct components of autophagy. By understanding how intracellular pathogens manipulate autophagy, we gain insight not only into bacterial pathogenesis but also host cell signaling and autophagolysosome maturation

Vasodilator-Stimulated Phosphoprotein Activity Is Required for <i>Coxiella burnetii</i> Growth in Human Macrophages

<div><p><i>Coxiella burnetii</i> is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and <i>C</i>. <i>burnetii</i> initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). <i>C</i>. <i>burnetii</i> manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during <i>C</i>. <i>burnetii</i> infection. Avirulent and virulent <i>C</i>. <i>burnetii</i> triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in <i>C</i>. <i>burnetii</i>-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.</p></div

VASP (S239E) expression prevents phagosome trafficking to the PV.

<p>THP-1 cells were transfected with wild type (WT) GFP-VASP (GFP-VASP), GFP-VASP (S157E), or GFP-VASP (S239E), then infected with <i>C</i>. <i>burnetii</i>. Cells were incubated with fluorescent beads (violet) overnight and processed for confocal microscopy at 72 hpi. DNA was stained with DAPI (blue). Bar, 10 μm. N = nucleus and * indicates the PV in micrographs. The scatter plot displays the percentage of beads present within PV in GFP-VASP-, GFP-VASP (S157E)-, or GFP-VASP (S239E)-expressing cells. At least 20 cells from randomly selected fields were used for quantification. The horizontal bar indicates average percentage. <b>*</b> indicates p < 0.0001 according to a Student’s <i>t</i> test comparing wild type- and mutant VASP-expressing cells. Bead-containing phagosomes were directed to the PV in GFP-VASP- and GFP-VASP (S157E)-expressing cells. However, GFP-VASP (S239E) expression impaired bead trafficking to the PV.</p

VASP activity is required for optimal PV formation.

<p>THP-1 cells were transfected with non-targeting (NT) or VASP-specific siRNA and knockdown confirmed by immunoblot. Cells were processed for confocal microscopy at 72 hpi. DNA was labeled with DAPI (blue), and CD63 (green) and <i>C</i>. <i>burnetii</i> (red) were detected with antibodies. Bar, 10 μm. The scatter plot displays PV diameter in NT or VASP siRNA-transfected cells. PV measurements were taken from at least 15 randomly selected fields. When multiple vacuoles were visible, the two largest vacuoles were measured in a single cell. The horizontal bar indicates average vacuole size. <b>*</b> indicates p < 0.0001 according to a Student’s <i>t</i> test comparing vacuole size in NT and VASP siRNA-transfected cells. VASP knockdown results in formation of smaller PV.</p

VASP activity is required for optimal <i>C</i>. <i>burnetii</i> intracellular replication.

<p>THP-1 cells were transfected with non-targeting (NT) or VASP-specific siRNA and harvested at 24–144 hpi. (A) Immunoblot analysis confirmed >80% knockdown of VASP production in VASP siRNA-transfected cells. (B) A viability assay demonstrated that no significant cell death occurred in VASP-depleted cells. (C) siRNA-transfected THP-1 cells were infected with <i>C</i>. <i>burnetii</i> expressing mCherry. Fluorescence was measured for 6 days after infection using a microplate reader (585/620 nm excitation/emission). (D) Bacterial genome equivalents were determined at 24 and 96 hpi using quantitative PCR. Each data point represents an average of seven replicates collected from two independent experiments, and error bars indicate standard deviation from the mean. <b>*</b> indicates p < 0.05, <b>**</b> indicates p < 0.0005, and *** indicates p < 0.0001 according to a Student’s <i>t</i> test comparing NT and VASP-silenced cells infected with <i>C</i>. <i>burnetii</i>-mCherry. Depletion of VASP significantly impairs <i>C</i>. <i>burnetii</i> intracellular growth.</p