An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses.

Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.

Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens.

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection in vivo remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.

Successful treatment of neurocysticercosis with albendazole desensitization.

BACKGROUND: Neurocysticercosis is a growing public health problem in the United States. Albendazole is a mainstay of medical therapy for neurocysticercosis, and here we present a case of hypersensitivity to albendazole leading to life-threatening disease progression. OBJECTIVE: To report the first successful albendazole desensitization protocol. METHODS: An oral albendazole 12-step desensitization protocol was developed, starting with 0.001 mg and progressing at 15 minutes intervals. Dosage for each subsequent step was as follows: 0.003 mg, 0.01 mg, 0.03 mg, 0.1 mg, 0.3 mg, 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300mg. RESULTS: The patient rapidly improved from a symptomatic standpoint, and repeat MRI showed a dramatic improvement in lesions. CONCLUSIONS: This successful desensitization protocol to albendazole can be of value to other patients with history suggestive of IgE-mediated allergy needing treatment for parasitic infections.

The balance between antiviral and antibacterial responses during M. tuberculosis infection is regulated by the ubiquitin ligase CBL.

As a first line of host defense, macrophages must be able to effectively sense and respond to diverse types of pathogens, and while a particular type of immune response may be beneficial in some circumstances, it can be detrimental in others. Upon infecting a macrophage, M. tuberculosis (Mtb) induces proinflammatory cytokines that activate antibacterial responses. Surprisingly, Mtb also triggers antiviral responses that actually hinder the ability of macrophages to control Mtb infection. The ubiquitin ligase CBL suppresses these antiviral responses and shifts macrophages toward a more antibacterial state during Mtb infection, however, the mechanisms by which CBL regulates immune signaling are unknown. We found that CBL controls responses to multiple stimuli and broadly suppresses the expression of antiviral effector genes. We then used mass-spectrometry to investigate potential CBL substrates and identified over 46,000 ubiquitylated peptides in Mtb-infected macrophages, as well as roughly 400 peptides with CBL-dependent ubiquitylation. We then performed genetic interaction analysis of CBL and its putative substrates, and identified the Fas associated factor 2 (FAF2) adapter protein as a key signaling molecule protein downstream of CBL. Together, these analyses identify thousands of new ubiquitin-mediated signaling events during the innate immune response and reveal an important new regulatory hub in this response.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes.

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model". However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.

Investigation of nosocomial SARS-CoV-2 transmission from two patients to healthcare workers identifies close contact but not airborne transmission events.

OBJECTIVE: To describe the pattern of transmission of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) during 2 nosocomial outbreaks of coronavirus disease 2019 (COVID-19) with regard to the possibility of airborne transmission. DESIGN: Contact investigations with active case finding were used to assess the pattern of spread from 2 COVID-19 index patients. SETTING: A community hospital and university medical center in the United States, in February and March, 2020, early in the COVID-19 pandemic. PATIENTS: Two index patients and 421 exposed healthcare workers. METHODS: Exposed healthcare workers (HCWs) were identified by analyzing the electronic medical record (EMR) and conducting active case finding in combination with structured interviews. Healthcare coworkers (HCWs) were tested for COVID-19 by obtaining oropharyngeal/nasopharyngeal specimens, and RT-PCR testing was used to detect SARS-CoV-2. RESULTS: Two separate index patients were admitted in February and March 2020, without initial suspicion for COVID-19 and without contact or droplet precautions in place; both patients underwent several aerosol-generating procedures in this context. In total, 421 HCWs were exposed in total, and the results of the case contact investigations identified 8 secondary infections in HCWs. In all 8 cases, the HCWs had close contact with the index patients without sufficient personal protective equipment. Importantly, despite multiple aerosol-generating procedures, there was no evidence of airborne transmission. CONCLUSION: These observations suggest that, at least in a healthcare setting, most SARS-CoV-2 transmission is likely to take place during close contact with infected patients through respiratory droplets, rather than by long-distance airborne transmission.

An M. tuberculosis Metabolic Enzyme Moonlights as an Anti-Inflammatory Effector Protein.

The mechanisms by which Mycobacterium tuberculosis evades host immunity remain enigmatic. In a recent study in the journal Nature, Wang et al. report that an enzyme acting in the bacterial cytoplasm to degrade lipids also acts as an effector protein in the host cell cytoplasm to suppress inflammation.

Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters.

We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.

A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation.

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here, we show that p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression.

Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression.

We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.

Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential.

Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.