scholarly journals Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

2020 ◽  
Author(s):  
Yong Lai ◽  
Gregory H. Babunovic ◽  
Liang Cui ◽  
Peter C. Dedon ◽  
John G. Doench ◽  
...  
Keyword(s):  
High Throughput ◽  
Mycobacterial Infection ◽  
Human Macrophage ◽  
Type I ◽  
Functional Genomic ◽  
Genetic Screens ◽  
Phagocytic Cells ◽  
Aryl Hydrocarbon ◽  
Tb Treatment ◽  
Mycobacterial Pathogenesis

SUMMARYExisting antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated high-throughput genetic screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Guérin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex host-pathogen interactions and serve to identify HDTs with the potential to improve TB treatment.

scholarly journals Functional Genomic Identification of Cadmium Resistance Genes from a High GC Clone Library by Coupling the Sanger and PacBio Sequencing Strategies

Genes ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 7
Author(s):  
Jinghao Chen ◽  
Chao Xing ◽  
Xin Zheng ◽  
Xiaofang Li
Keyword(s):  
High Throughput ◽  
Resistance Genes ◽  
Sanger Sequencing ◽  
Genomic Library ◽  
Full Length ◽  
Host Cells ◽  
Full Genome Sequence ◽  
Functional Screening ◽  
Functional Genomic ◽  
Pacbio Sequencing

Functional (meta) genomics allows the high-throughput identification of functional genes in a premise-free way. However, it is still difficult to perform Sanger sequencing for high GC DNA templates, which hinders the functional genomic exploration of a high GC genomic library. Here, we developed a procedure to resolve this problem by coupling the Sanger and PacBio sequencing strategies. Identification of cadmium (Cd) resistance genes from a small-insert high GC genomic library was performed to test the procedure. The library was generated from a high GC (75.35%) bacterial genome. Nineteen clones that conferred Cd resistance to Escherichia coli subject to Sanger sequencing directly. The positive clones were in parallel subject to in vivo amplification in host cells, from which recombinant plasmids were extracted and linearized by selected restriction endonucleases. PacBio sequencing was performed to obtain the full-length sequences. As the identities, partial sequences from Sanger sequencing were aligned to the full-length sequences from PacBio sequencing, which led to the identification of seven unique full-length sequences. The unique sequences were further aligned to the full genome sequence of the source strain. Functional screening showed that the identified positive clones were all able to improve Cd resistance of the host cells. The functional genomic procedure developed here couples the Sanger and PacBio sequencing methods and overcomes the difficulties in PCR approaches for high GC DNA. The procedure can be a promising option for the high-throughput sequencing of functional genomic libraries, and realize a cost-effective and time-efficient identification of the positive clones, particularly for high GC genetic materials.

scholarly journals Lymphocytes are detrimental during the early innate immune response against Listeria monocytogenes

2006 ◽  
Vol 203 (4) ◽  
pp. 933-940 ◽  
Author(s):  
Javier A. Carrero ◽  
Boris Calderon ◽  
Emil R. Unanue
Keyword(s):  
Immune Response ◽  
Listeria Monocytogenes ◽  
Innate Immune Response ◽  
Early Stage ◽  
Bacterial Pathogen ◽  
Interleukin 10 ◽  
Innate Immune ◽  
Type I ◽  
Phagocytic Cells ◽  
Immunodeficient Mice

Mice deficient in lymphocytes are more resistant than normal mice to Listeria monocytogenes infection during the early innate immune response. This paradox remains unresolved: lymphocytes are required for sterilizing immunity, but their presence during the early stage of the infection is not an asset and may even be detrimental. We found that lymphocyte-deficient mice, which showed limited apoptosis in infected organs, were resistant during the first four days of infection but became susceptible when engrafted with lymphocytes. Engraftment with lymphocytes from type I interferon receptor–deficient (IFN-αβR−/−) mice, which had reduced apoptosis, did not confer increased susceptibility to infection, even when the phagocytes were IFN-αβR+/+. The attenuation of innate immunity was due, in part, to the production of the antiinflammatory cytokine interleukin 10 by phagocytic cells after the apoptotic phase of the infection. Thus, immunodeficient mice were more resistant relative to normal mice because the latter went through a stage of lymphocyte apoptosis that was detrimental to the innate immune response. This is an example of a bacterial pathogen creating a cascade of events that leads to a permissive infective niche early during infection.

scholarly journals Targeted High-Throughput Sequencing Identifies Mutations in atlastin-1 as a Cause of Hereditary Sensory Neuropathy Type I

2011 ◽  
Vol 88 (1) ◽  
pp. 99-105 ◽  
Author(s):  
Christian Guelly ◽  
Peng-Peng Zhu ◽  
Lea Leonardis ◽  
Lea Papić ◽  
Janez Zidar ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sensory Neuropathy ◽  
Type I ◽  
Hereditary Sensory Neuropathy

scholarly journals Significant Growth by Rickettsia Species within Human Macrophage-Like Cells Is a Phenotype Correlated with the Ability to Cause Disease in Mammals

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 228
Author(s):  
M. Nathan Kristof ◽  
Paige E. Allen ◽  
Lane D. Yutzy ◽  
Brandon Thibodaux ◽  
Christopher D. Paddock ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Spotted Fever ◽  
Growth Characteristic ◽  
Human Macrophage ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
Rocky Mountain Spotted Fever ◽  
Phagocytic Cells ◽  
Pathogenic Potential ◽  
Rickettsia Species

Rickettsia are significant sources of tick-borne diseases in humans worldwide. In North America, two species in the spotted fever group of Rickettsia have been conclusively associated with disease of humans: Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, and Rickettsia parkeri, the cause of R. parkeri rickettsiosis. Previous work in our lab demonstrated non-endothelial parasitism by another pathogenic SFG Rickettsia species, Rickettsia conorii, within THP-1-derived macrophages, and we have hypothesized that this growth characteristic may be an underappreciated aspect of rickettsial pathogenesis in mammalian hosts. In this work, we demonstrated that multiple other recognized human pathogenic species of Rickettsia, including R. rickettsii, R. parkeri, Rickettsia africae, and Rickettsiaakari can grow within target endothelial cells as well as within PMA-differentiated THP-1 cells. In contrast, Rickettsia bellii, a Rickettsia species not associated with disease of humans, and R. rickettsii strain Iowa, an avirulent derivative of pathogenic R. rickettsii, could invade both cell types but proliferate only within endothelial cells. Further analysis revealed that similar to previous studies on R. conorii, other recognized pathogenic Rickettsia species could grow within the cytosol of THP-1-derived macrophages and avoided localization with two different markers of lysosomal compartments; LAMP-2 and cathepsin D. R. bellii, on the other hand, demonstrated significant co-localization with lysosomal compartments. Collectively, these findings suggest that the ability of pathogenic rickettsial species to establish a niche within macrophage-like cells could be an important factor in their ability to cause disease in mammals. These findings also suggest that analysis of growth within mammalian phagocytic cells may be useful to predict the pathogenic potential of newly isolated and identified Rickettsia species.

Effects of Berberine on Expression of LOX-1 and SR-BI in Human Macrophage-Derived Foam Cells Induced by ox-LDL

2010 ◽  
Vol 38 (06) ◽  
pp. 1161-1169 ◽  
Author(s):  
Siming Guan ◽  
Bin Wang ◽  
Wei Li ◽  
Jinghuan Guan ◽  
Xin Fang
Keyword(s):  
Scavenger Receptor ◽  
Ldl Receptor ◽  
Foam Cells ◽  
Time Dependent ◽  
Human Macrophage ◽  
Type I ◽  
Dependent Manner ◽  
Atp Binding Cassette Transporter ◽  
Abca1 Expression ◽  
Class B

This study investigates the effects of beriberine on the expression of lectin-like ox-LDL receptor-1 (LOX-1), scavenger receptor A (SR-A), SR class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in human macrophage-derived foam cells induced by ox-LDL. Different concentrations of Berberine were co-cultured with THP-1 derived foam cells. The mRNA and protein expressions of LOX-1, SR-A, SR-BI and ABCA1 were determined by RT-PCR and Western blot analysis, respectively. Ox-LDL significantly increased the expression of LOX-1 and inhibited the expression of SR-BI in a dose- and time-dependent manner. Berberine significantly inhibited the effects of ox-LDL in a dose- and time-dependent manner. Moreover, ox-LDL significantly promoted ABCA1 expression. However, berberine had no effect on SR-A or ABCA1 expression. Berberine can inhibit the expression of LOX-1 and promote the expression of SR-BI in macrophage-derived foam cells. Therefore, berberine could be used to treat atherosclerotic diseases.

scholarly journals Mycobacterial infection of precision cut lung slices reveals that the type 1 interferon pathway is locally induced by Mycobacterium bovis but not M. tuberculosis in different cattle breeds

2021 ◽  
Author(s):  
Aude Remot ◽  
Florence Carreras ◽  
Anthony Coupe ◽  
Emilie Doz-Deblauwe ◽  
Maria-Laura Boschiroli ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Animal Health ◽  
Mycobacterial Infection ◽  
Developed Countries ◽  
Host Cells ◽  
Economic Losses ◽  
Reference Laboratory ◽  
Type I ◽  
Cattle Breeds ◽  
Interferon Pathway

Tuberculosis exacts a terrible toll on human and animal health. While Mycobacterium tuberculosis (Mtb) is restricted to humans, Mycobacterium bovis (Mb) is present in a large range of mammalian hosts. In cattle, bovine TB (bTB) is a notifiable disease responsible for important economic losses in developed countries and underestimated zoonosis in the developing world. Early interactions that take place between mycobacteria and the lung tissue early after aerosol infection govern the outcome of the disease. In cattle, these early steps remain poorly characterized. The precision-cut lung slice (PCLS) model preserves the structure and cell diversity of the lung. We developed this model in cattle in order to study the early lung response to mycobacterial infection. In situ imaging of PCLS infected with fluorescent Mb revealed bacilli in the alveolar compartment, adjacent or inside alveolar macrophages (AMPs) and in close contact with pneumocytes. We analyzed the global transcriptional lung inflammation signature following infection of PCLS with Mb and Mtb in two French beef breeds: Blonde d'Aquitaine and Charolaise. Whereas lungs from the Blonde d'Aquitaine produced high levels of mediators of neutrophil and monocyte recruitment in response to infection, such signatures were not observed in the Charolaise in our study. In the Blonde d'Aquitaine lung, whereas the inflammatory response was highly induced by two Mb strains, AF2122 isolated from cattle in the UK and Mb3601 circulating in France, the response against two Mtb strains, H37Rv the reference laboratory strain and BTB1558 isolated from zebu in Ethiopia, was very low. Strikingly, the type I interferon pathway was only induced by Mb but not Mtb strains indicating that this pathway may be involved in mycobacterial virulence and host tropism. Hence, the PCLS model in cattle is a valuable tool to deepen our understanding of early interactions between lung host cells and mycobacteria. It revealed striking differences between cattle breeds and mycobacterial strains. This model could help deciphering biomarkers of resistance versus susceptibility to bTB in cattle as such information is still critically needed for bovine genetic selection programs and would greatly help the global effort to eradicate bTB.

scholarly journals Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE

2021 ◽  
Author(s):  
Oliver M. Crook ◽  
Colin T. R. Davies ◽  
Laurent Gatto ◽  
Paul D.W. Kirk ◽  
Kathryn S. Lilley
Keyword(s):  
Steady State ◽  
High Throughput ◽  
Statistical Power ◽  
Subcellular Localisation ◽  
Type I ◽  
Disease States ◽  
Proteomic Data ◽  
Rational Drug ◽  
Context Specific ◽  
Type Ii Errors

AbstractThe steady-state localisation of proteins provides vital insight into their function. These localisations are context specific with proteins translocating between different sub-cellular niches upon perturbation of the subcellular environment. Differential localisation provides a step towards mechanistic insight of subcellular protein dynamics. Aberrant localisation has been implicated in a number of pathologies, thus differential localisation may help characterise disease states and facilitate rational drug discovery by suggesting novel targets. High-accuracy high-throughput mass spectrometry-based methods now exist to map the steady-state localisation and re-localisation of proteins. Here, we propose a principled Bayesian approach, BANDLE, that uses these data to compute the probability that a protein differentially localises upon cellular perturbation, as well quantifying the uncertainty in these estimates. Furthermore, BANDLE allows information to be shared across spatial proteomics datasets to improve statistical power. Extensive simulation studies demonstrate that BANDLE reduces the number of both type I and type II errors compared to existing approaches. Application of BANDLE to datasets studying EGF stimulation and AP-4 dependent localisation recovers well studied translocations, using only two-thirds of the provided data. Moreover, we implicate TMEM199 with AP-4 dependent localisation. In an application to cytomegalovirus infection, we obtain novel insights into the rewiring of the host proteome. Integration of high-throughput transcriptomic and proteomic data, along with degradation assays, acetylation experiments and a cytomegalovirus interactome allows us to provide the functional context of these data.

scholarly journals 3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role

2020 ◽  
Vol 21 (24) ◽  
pp. 9722
Author(s):  
Nicolò Baranzini ◽  
Laura Pulze ◽  
Marcella Reguzzoni ◽  
Rossella Roncoroni ◽  
Viviana Teresa Orlandi ◽  
...  
Keyword(s):  
Light Transmission ◽  
Three Dimensional ◽  
Lipoteichoic Acid ◽  
Type I ◽  
Phagocytic Cells ◽  
In Vivo Experiments ◽  
Macrophage Phagocytosis ◽  
Bacterial Aggregates

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech’s innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health.

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