Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly ...understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.
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•The effector SopD2 blocks delivery of endocytic cargo to lysosomes•SopD2 interacts directly with host GTPase Rab7•SopD2’s N terminus mediates both trafficking suppression and interaction with Rab7•SopD2 impairs Rab7’s ability to bind cognate effectors RILP and FYCO1
Intracellular pathogens can evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation. D’Costa et al. show that the effector SopD2 mediates this process in Salmonella infection by binding the host regulatory GTPase Rab7 and impairing its ability to recruit dynein- and kinesin-binding effectors RILP and FYCO1.
An adult with hypomorphic ZAP-70 deficiency due to de novo donor splice site mutation manifested autoimmunity, infections especially with DNA-based viruses and lymphoproliferation. Morpholino ...interference of mutant splice site increased expression of wild-type protein, significantly improving T cell responses.
Summary
The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella‐containing vacuole (SCV), in host cells. Here we demonstrate ...that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B+ tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1+ tubules (the Salmonella‐induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin‐1. Here we find that Arl8B is required for kinesin‐1 recruitment to SCVs. We have previously shown that SCVs undergo centrifugal movement to the cell periphery at 24 h post infection and undergo cell‐to‐cell transfer to infect neighbouring cells, and that both phenotypes require kinesin‐1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell‐to‐cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co‐opted by S. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.
Clostridioides difficile is a major cause of health care-associated diarrhea. Severity ranges from mild to life-threatening, but this variability remains poorly understood. Microbiologic diagnosis of ...C. difficile infection (CDI) is straightforward but offers little insight into the patient's prognosis or into pathophysiologic determinants of clinical trajectory. The aim of this study was to discover host-derived, CDI-specific fecal biomarkers involved in disease severity. Subjects without and with CDI diarrhea were recruited. CDI severity was based on Infectious Diseases Society of America/Society for Healthcare Epidemiology of America criteria. We developed a liquid chromatography tandem mass spectrometry approach to identify host-derived protein biomarkers from stool and applied it to diagnostic samples for cohort-wise comparison (CDI-negative vs. nonsevere CDI vs. severe CDI). Selected biomarkers were orthogonally confirmed and subsequently verified in a CDI mouse model. We identified a protein signature from stool, consisting of alpha-2-macroglobulin (A2MG), matrix metalloproteinase-7 (MMP-7), and alpha-1-antitrypsin (A1AT), that not only discriminates CDI-positive samples from non-CDI ones but also is potentially associated with disease severity. In the mouse model, this signature with the murine homologs of the corresponding proteins was also identified. A2MG, MMP-7, and A1AT serve as biomarkers in patients with CDI and define novel components of the host response that may determine disease severity.
Tissue-resident alveolar macrophages are important for lung homeostasis and innate immunity. They originate from fetal monocytes and are maintained in the lung through self-renewal or may arise from ...recruited circulating monocytes. Disturbance of alveolar macrophage function, their numbers or loss of GM-CSF signaling can all underlie pulmonary alveolar proteinosis (PAP). Here, we report eight patients (aged 8–21 years) from four kindreds and three ancestries suffering from recurrent pneumonia, progressive interstitial fibrosis, and PAP. All patients are deficient for the monocyte-chemoattractant protein-1 (CCL-2) receptor 2 (CCR2). Five patients are homozygous and three are compound heterozygous for private or rare CCR2 variants. The variants are loss-of-function for Ca2 +-release and cell migration upon CCL-2 stimulation, when tested by overexpression in monocytic THP-1 cells. Moreover, CCL-2-dependent migration and Ca2+-release of the patients’ monocytes are abolished, while their plasma CCL-2 levels are elevated. Yet, CCR2-deficient cells respond normally to GM-CSF. In addition, the counts, frequencies, and transcriptomes of blood lymphoid and myeloid subsets are normal. Mice deficient for CCR2 display alveolar septal wall thickening and increased number of alveolar type II cells producing surfactant protein C causing increased lung tissue hysteresis. CCR2-deficient mice are vulnerable to mycobacteria, with impaired attraction of blood monocytes to infected tissues. These findings suggest that human CCR2 is essential for the recruitment of monocytes to both pulmonary alveoli and tissues infected with mycobacteria.
Loss of function mutations in human Caspase Recruitment Domain Containing Protein 9 (CARD9) cause invasive fungal infections, particularly of the central nervous system (CNS) with the fungus Candida ...albicans. CNS candidiasis is modeled in mice by intravenous injection to disseminate yeast cells throughout the body. However, at the challenge doses currently used, Card9-/- mice die from acute sepsis, making it difficult to relate findings to deep organ invasive candidiasis in humans with CARD9-deficiency. These human infections are often late-onset or chronic in nature and are not accompanied by acute sepsis. There is therefore an unmet need for the development of a model to study the pathogenesis of chronic tissue infections as well as the immune mechanisms underlying them. The recently identified p.Y91H mutation in CARD9 causes CNS candidiasis, however the immunological mechanisms by which p.Y91H leads to this disease remain to be fully characterized. We present a novel model of chronic invasive candidiasis in CARD9-deficiency with late onset chronology of disease symptoms. We show that p.Y91H knock-in mice (Y91HKI) phenocopy Card9-/- mice at the level of survival, dissemination, and tissue fungal burden but have divergent severity of neurological symptoms late in infection. Studies using the candidemia model of disseminated candidiasis have shown a granulopoietic response in the bone marrow and demonstrated that bone marrow hematopoietic stem and progenitor cells (HSPC) can proliferate in response to direct C. albicans challenge. We show that Card9-/- mice mount a robust, progressive, and transient emergency myelopoiesis response over the course of asymptomatic infection. Beta-glucan challenge in Card9-/- mice failed to induce HSPC expansion in vivo and Card9-/- BMDM from these mice showed augmented cytokine responses to C. albicans challenge in vitro. Together, our results show that Card9-/- mice mount a progressive emergency hematopoiesis response and suggest a role for CARD9 in fungal-induced trained immunity. By extending the chronology of disease, our work provides a new temporal framework for investigation of invasive candidiasis, and development of intervention strategies, by defining distinct phases of fungal growth, immune response, and disease in the context of CARD9 primary immunodeficiency.
Primary immunodeficiencies represent naturally occurring experimental models to decipher human immunobiology. We report a patient with combined immunodeficiency, marked by recurrent respiratory tract ...and DNA-based viral infections, hypogammaglobulinemia, and panlymphopenia. He also developed moderate neutropenia but without prototypical pyogenic infections. Using whole-exome sequencing, we identified a homozygous mutation in the inducible T cell costimulator ligand gene (
; c.657C>G; p.N219K). Whereas WT ICOSL is expressed at the cell surface, the ICOSL
mutation abrogates surface localization: mutant protein is retained in the endoplasmic reticulum/Golgi apparatus, which is predicted to result from deleterious conformational and biochemical changes. ICOSL
diminished B cell costimulation of T cells, providing a compelling basis for the observed defect in antibody and memory B cell generation. Interestingly, ICOSL
also impaired migration of lymphocytes and neutrophils across endothelial cells, which normally express ICOSL. These defects likely contributed to the altered adaptive immunity and neutropenia observed in the patient, respectively. Our study identifies human
deficiency as a novel cause of a combined immunodeficiency.
We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent ...kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.
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•Inherited human CCR2 deficiency impairs pulmonary recruitment of monocytes•Insufficient attraction of monocytes reduces the counts of alveolar macrophages•Reduced counts of human alveolar macrophages underlie chronic lung disease•Inherited human CCR2 deficiency also impairs protective immunity to mycobacteria
While the chemokine receptor CCR2 has been understood as a key factor in monocyte homing in mice, the study of a naturally occurring CCR2 genetic deficiency in patients suggests that, in humans, this chemokine receptor is of particular importance for the recruitment and maturation of macrophages in the lungs.
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