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Published on: August 5, 2016
Blocking the expression of syntaxin 4 interferes with initial phagocytosis of Brucella melitensis in macrophages
Alfredo Castañeda-Ramírez1, Diana González-Rodríguez1, J Aide Hernández-Pineda1
1Laboratorio de Microbiología Molecular, Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Av. Universidad 3000, Circuito Exterior s/n, CU, Distrito Federal 04510, México (Castañeda-Ramírez, González-Rodríguez, Hernández-Pineda, Verdugo-Rodríguez); Departamento de Zootecnia, Universidad Autónoma Chapingo, Texcoco, México (Castañeda-Ramírez).
Abstract:
Brucella melitensis is the Brucella species most frequently associated with brucellosis in humans. It is also the causative agent of the disease in goats and other ruminants. Although significant aspects of the pathogenesis of infection by this intracellular pathogen have been clarified, several events during invasion of host cells remain to be elucidated. In this study, infections of human macrophages from the THP-1 monocyte cell line were conducted with B. melitensis Bm133 wild-type strain and a strain of Salmonella serovar Enteritidis as a control. A multiplicity of infection of 100 was used in trials focused on defining the relative expression of syntaxin 4 (STX4), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, in the early events of phagocytosis (at 15, 30, 45, and 60 min). Immunoblot assays were also done to visualize expression of the protein in cells infected with either bacterial strain. The expression of STX4 was not significantly different in cells infected with B. melitensis strain Bm133 compared to that observed in cells infected with S. Enteritidis. When the expression of STX4 mRNA was inhibited with short or small interfering, or silencing, RNA in the THP-1 cells, the survival of B. melitensis was significantly reduced at time 0, when gentamicin treatment of cultures was begun (after 1 h of phagocytosis), and also at 2 h and 12 h after infection.
Insights
Brucella melitensis survival in human macrophages depends on syntaxin 4 (STX4). Inhibiting STX4 significantly reduced B. melitensis survival, highlighting its role in early infection stages.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Brucella melitensis causes human brucellosis and infects ruminants.
- Pathogenesis of B. melitensis intracellular infection requires further elucidation, particularly host cell invasion events.
Purpose of the Study:
- To investigate the role of syntaxin 4 (STX4) in the early stages of human macrophage infection by B. melitensis.
- To determine if STX4 expression is altered during B. melitensis phagocytosis.
Main Methods:
- Infection of THP-1 human macrophages with B. melitensis and Salmonella Enteritidis.
- Analysis of syntaxin 4 (STX4) expression using immunoblot assays and mRNA analysis.
- Silencing of STX4 mRNA using small interfering RNA (siRNA) to assess its impact on bacterial survival.
Main Results:
- STX4 expression levels were comparable between B. melitensis and S. Enteritidis infected cells.
- Inhibition of STX4 mRNA expression significantly reduced B. melitensis survival post-infection.
- STX4 plays a crucial role in the survival of B. melitensis within human macrophages.
Conclusions:
- Syntaxin 4 (STX4) is essential for the intracellular survival of Brucella melitensis.
- Targeting STX4 could be a potential strategy to combat B. melitensis infections.
