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EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.

ABSTRACT: Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.

SUBMITTER: Garces A

PROVIDER: S-EPMC2891827 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.

Garces Alejandra A Atmakuri Krishnamohan K Chase Michael R MR Woodworth Joshua S JS Krastins Bryan B Rothchild Alissa C AC Ramsdell Talia L TL Lopez Mary F MF Behar Samuel M SM Sarracino David A DA Fortune Sarah M SM

PLoS pathogens 20100624 6

Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishi ...[more]

PMID: 20585630

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Project description:CHY1 is a zinc finger protein unique to microorganisms that was found to regulate polarized tip growth in Fusarium graminearum, an important pathogen of wheat and barley. To further characterize its functions, in this study we identified CHY1-interacting proteins by affinity purification and selected UDP-galactofuranose (Galf) mutase (UGMA) for detailed characterization, because UGMA and UDP-Galf are unique to fungi and bacteria and absent in plants and animals. The interaction between CHY1 and UGMA was confirmed by yeast two-hybrid assays. Deletion of UGMA in F. graminearum resulted in significant defects in vegetative growth, reproduction, cell wall integrity, and pathogenicity. Infection with the ΔugmA mutant was restricted to the inoculated floret, and no vomitoxin was detected in kernels inoculated with the ΔugmA strain. Compared to the wild type, the ΔugmA mutant produced wide, highly branched hyphae with thick walls, as visualized by transmission electron microscopy. UGMA tagged with green fluorescent protein (GFP) mainly localized to the cytoplasm, consistent with the synthesis of Galf in the cytoplasm. The Δchy1 mutant was more sensitive, while the ΔugmA mutant was more tolerant, to cell wall-degrading enzymes. The growth of the ΔugmA mutant nearly ceased upon caspofungin treatment. More interestingly, nocodazole treatment of the ΔugmA strain attenuated its highly branched morphology, while caspofungin inhibited the degree of the twisted Δchy1 mycelia, indicating that CHY1 and UGMA probably have opposite effects on cell wall architecture. In conclusion, UGMA is an important pathogenic factor that is specific to fungi and bacteria and required for cell wall architecture, radial growth, and caspofungin tolerance, and it appears to be a promising target for antifungal agent development. IMPORTANCE The long-term use of chemical pesticides has had increasingly negative impacts on the ecological environment and human health. Low-toxicity, high-efficiency and environmentally friendly alternative pesticides are of great significance for maintaining the sustainable development of agriculture and human and environmental health. Using fungus- or microbe-specific genes as candidate targets provides a good foundation for the development of low-toxicity, environmentally friendly pesticides. In this study, we characterized a fungus- and bacterium-specific UDP-galactopyranose mutase gene, ugmA, that contributes to the synthesis of the cell wall component Galf and is required for vegetative growth, cell wall integrity, deoxynivalenol (DON) production, and pathogenicity in F. graminearum. The ugmA deletion mutant exhibited increased sensitivity to caspofungin. These results demonstrate the functional importance of UGMA in F. graminearum, and its absence from mammals and higher plants constitutes a considerable advantage as a low-toxicity target for the development of new anti-Fusarium agents.

Dataset Information

EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.

Publications

EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.

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