scholarly journals Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection

2020 ◽  
Vol 8 (10) ◽  
pp. 1571
Author(s):  
Ida L. Phillips ◽  
Jamie L. Everman ◽  
Luiz E. Bermudez ◽  
Lia Danelishvili
Keyword(s):  
Virulence Factors ◽  
Mycobacterium Avium ◽  
Screening Tool ◽  
Gene Knockout ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Acanthamoeba Castellanii ◽  
Mammalian Host ◽  
Intracellular Growth ◽  
Macrophage Infection ◽  
Selection Of

The high prevalence of Johne’s disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

scholarly journals Thioridazine Is an Efflux Pump Inhibitor in Mycobacterium avium Complex but of Limited Clinical Relevance

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Mike Marvin Ruth ◽  
Lian J. Pennings ◽  
Valerie A. C. M. Koeken ◽  
Jodie A. Schildkraut ◽  
Aria Hashemi ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Mycobacterium Avium Complex ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Plasma Concentrations ◽  
Intracellular Growth ◽  
Macrophage Infection ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Time Kill

ABSTRACT Treatment of Mycobacterium avium complex pulmonary disease (MAC-PD) is challenging partly due to high efflux pump expression. Thioridazine might block these efflux pumps. We explore the efficacy of thioridazine against M. avium isolates using MICs, time-kill combination assays, ex vivo macrophage infection assays, and efflux assays. Thioridazine is bactericidal against M. avium, inhibits intracellular growth at 2× MIC, and blocks ethidium bromide efflux. However, its toxicity and low plasma concentrations make it unlikely to add efficacy to MAC-PD therapy.

scholarly journals De Novo Purine Biosynthesis Is Required for Intracellular Growth of Staphylococcus aureus and for the Hypervirulence Phenotype of a purR Mutant

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Mariya I. Goncheva ◽  
Ronald S. Flannagan ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Metabolic Regulation ◽  
De Novo ◽  
Purine Biosynthesis ◽  
Mammalian Host ◽  
Cell Binding ◽  
Intracellular Growth ◽  
Content Type ◽  
De Novo Purine Biosynthesis

ABSTRACT Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.

scholarly journals Mycobacterium avium Subspecies Paratuberculosis and Crohn’s Regional Ileitis: How Strong is Association?

2011 ◽  
Vol 3 (02) ◽  
pp. 069-074 ◽  
Author(s):  
Sarman Singh ◽  
Krishnamoorthy Gopinath
Keyword(s):  
Mycobacterium Avium ◽  
External Factor ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Point Of View ◽  
Etiological Agent ◽  
Regional Ileitis ◽  
Genetic Traits ◽  
Human Genes ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Selection Of

ABSTRACT Mycobacterium avium subsp. paratuberculosis (MAP) is a well-established etiological agent of Johne’s disease in animals. In humans, similar clinical condition, first described by Crohn as regional ileitis in 1932, now known as Crohn’s diseases (CD), has also been associated with this mycobacterial species. However, there are two schools of thoughts, one favoring MAP as its etiological agent while the second considers it as an immune-inflammatory condition triggered by an external factor. Onset of CD requires a series of events including predisposition of certain inherited genetic traits, associated environmental stimuli, and immune-inflammatory response. A combination of these factors probably leads to this disease. Recently, some human genes have also been identified which regulate ability to respond appropriately to the external factors. Added to these factors are concerns about the selection of clinical specimens and poor adherence to laboratory quality controls. The literature is full of contradictory findings, but there a lack of uniformity in the materials and methods used by many of these researchers. In this review, we provide our perspective under above circumstances and give our point of view which may open a platform for debate regarding the MAP as the etiological agent of human CD.

scholarly journals Macrophage Proteome Analysis at Different Stages of Mycobacterium avium Subspecies paratuberculosis Infection Reveals a Mechanism of Pathogen Dissemination

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 20
Author(s):  
Ida L. Phillips ◽  
Lia Danelishvili ◽  
Luiz E. Bermudez
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Global Analysis ◽  
Natural Infection ◽  
The Body ◽  
Enteric Infection ◽  
Phagocytic Cells ◽  
Macrophage Infection

Johne’s disease is a chronic and usually fatal enteric infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) and is responsible for hundreds of millions of dollars in losses for the agricultural industry. Natural infection typically begins with bacterial uptake and translocation through the epithelium of the small intestine, followed by ingestion by tissue macrophages and dissemination via the lymphatic or blood system throughout the body. To gain insights into the host responses and adaptation of MAP within phagocytic cells, we utilized the previously developed cell culture passage model, and mass spectrometric-based quantitative proteomic approach. Using the cell culture system, which mimics an in vivo interaction of MAP with intestinal epithelium and tissue macrophages, bacteria were passed through the bovine epithelial cells and, subsequently, used for macrophage infection (termed indirect infection), while uninfected cells and macrophage infection initiated with the culture grown bacteria (termed direct infection) served as controls. Approximately 3900 proteins were identified across all studied groups. The comparison within the subset of proteins that showed synthesis for more than two-fold in the direct infection over the uninfected control revealed an enrichment for the pro-inflammatory pathways such as the NF-κB and cytokine/chemokine signaling, positive regulation of defense response, cell activation involved in the immune response and adaptive immune system. While these responses were absent in the indirect infection, cellular pathways such as cell cycle, healing, regulation of cell adhesion, ensemble of core extracellular matrix proteins, cell surface integrins and proteins mediating the integrin signaling were remarkably high within the indirect infection. In addition to global analysis of the macrophage proteome, we further validated the proteomics data and confirmed that MAP passage through epithelial cells modulates the expression and signaling of integrins in phagocytes. In this study, we demonstrate that predominant expression of integrins in the indirectly infected macrophages allows phagocytic cells to initiate stronger binding and efficient translocation through the endothelial cells, suggesting the important role of integrins in the spread of MAP infection.

Epidemiological and genetic markers, virulence factors and intracellular growth of Mycobacterium avium in AIDS

1992 ◽  
Vol 143 (4) ◽  
pp. 423-430 ◽  
Author(s):  
J.J. McFadden ◽  
Z.M. Kunze ◽  
F. Portaels ◽  
V. Labrousse ◽  
N. Rastogi
Keyword(s):  
Genetic Markers ◽  
Virulence Factors ◽  
Mycobacterium Avium ◽  
Intracellular Growth

scholarly journals The novel Dbl homology/BAR domain protein, MsgA, of Talaromyces marneffei regulates yeast morphogenesis during growth inside host cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Harshini Weerasinghe ◽  
Hayley E. Bugeja ◽  
Alex Andrianopoulos
Keyword(s):  
Pathogenic Fungi ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Mammalian Host ◽  
Host Immune System ◽  
Nucleotide Exchange ◽  
Phagocytic Cells ◽  
Macrophage Infection ◽  
Bar Domain ◽  
Talaromyces Marneffei

AbstractMicrobial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marneffei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifies a temperature independent pathway in T. marneffei that controls intracellular yeast morphogenesis.

scholarly journals Galleria mellonella as a screening tool to study virulence factors of Aspergillus fumigatus

Virulence ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 818-834
Author(s):  
Marie-Fleur Durieux ◽  
Élise Melloul ◽  
Sana Jemel ◽  
Lolita Roisin ◽  
Marie-Laure Dardé ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Virulence Factors ◽  
Screening Tool ◽  
Galleria Mellonella
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