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Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection.

ABSTRACT: The foodborne pathogen Listeria monocytogenes employs a number of virulence determinants including metalloproteases to infect hosts. Here for the first time, we identified an M29 family aminopeptidase T (encoded by lmo1603) from L. monocytogenes that possesses a typical feature to catalyze the cleavage of amino acids from peptide substrates, with a preference for arginine. The purified recombinant Lmo1603 was activated by Fe(3+), Zn(2+) and Mn(2+), but strongly stimulated by Co(2+), indicating that Lmo1603 is a cobalt-dependent aminopeptidase. Single mutation at any of the Glu216, Glu281, His308, Tyr315, His327, and Asp329 completely abolished the enzymatic activity of Lmo1603. More importantly, we showed that Lmo1603 was mainly involved in Listeria infection, but not required for growth in rich laboratory medium and minimal defined medium. Disruption of Lmo1603 resulted in almost complete attenuation of Listeria virulence in a mouse infection model. In addition, we demonstrated that Lmo1603 was mainly localized in the bacterial cytosol and required for invasion and survival inside human epithelial cells and murine macrophages. We conclude that Lmo1603 encodes a functional aminopeptidase T of M29 family, which acts as a novel intracellular virulence factor essential in the successful establishment of L. monocytogenes infections in a mouse model.

SUBMITTER: Cheng C

PROVIDER: S-EPMC4661694 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection.

Cheng Changyong C Wang Xiaowen X Dong Zhimei Z Shao Chunyan C Yang Yongchun Y Fang Weihuan W Fang Chun C Wang Hang H Yang Menghua M Jiang Lingli L Zhou Xiangyang X Song Houhui H

Scientific reports 20151127

The foodborne pathogen Listeria monocytogenes employs a number of virulence determinants including metalloproteases to infect hosts. Here for the first time, we identified an M29 family aminopeptidase T (encoded by lmo1603) from L. monocytogenes that possesses a typical feature to catalyze the cleavage of amino acids from peptide substrates, with a preference for arginine. The purified recombinant Lmo1603 was activated by Fe(3+), Zn(2+) and Mn(2+), but strongly stimulated by Co(2+), indicating t ...[more]

PMID: 26610705

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Project description:Cerebral listeriosis is characterized by neuronal apoptosis and microglial cell activation, but little is known about the bacterial virulence factors involved in this process and how bacterial dissemination is controlled. Here, we show that the cellular target of Listeria monocytogenes (LM) in murine hippocampal cultures is microglia rather than neurons or other glial cells, which are rarely infected. This in vitro model served to demonstrate that infected microglial cells release a soluble factor to the medium responsible for neuronal apoptosis. We investigated the production of this factor in a well-established murine microglia cell model BV2 cells, and compared with J-774 macrophage cells after infection with different LM bacterial mutants. Our purpose was to study in both cell types parameters such as the listericidal capacities, pro-inflammatory cytokines released, and bacterial factors involved in the intracellular cycle. Our data reveal that microglia shows unique features to handle a LM infection. Microglia is three times more permissive for LM intracellular growth than murine macrophages; while producing five times higher levels of pro-inflammatory cytokines IL-6, MCP-1 and TNF-α than macrophages. Using different LM mutants (i.e., LMWT, LM∆LLO, LM∆ActA or LM∆plcB) we conclude that LLO and ActA are not involved in LM proliferation within microglia cells, while they are required for survival within macrophages. Moreover, ActA controls TNF-α production, a cytokine involved in neural apoptosis. Transcriptional differential response of microglia infected with different LM mutants reflected ActA controls the TNF-a signaling pathway through out molecules and chemokines involved in this pathway such as NFkβ, MAPK-1, Ccl2, Ccl3, Ccl4 and CxCl10. The project implies the analysis of two replicates from three different conditions, in total 6 samples: microglia cells non-infected, microglia cells infected with Listeria monocytogenes (LM) wild type (LLO+), microglia cells infected with LM deficient in listeriolysin O (LLO-) and microglia cells infected with LM deficient in ActA (1942). References samples were microglia cells non-infected (NT). Infection with LM was performed for 1 hour, followed by 24 hours in medium with antibiotics to avoid bacterial extracellular growth. Gene expression analysis was performed using Partek Genomics Suite software (version 6.11.0801; Partek). GeneChip data were filtered to remove those probe sets with an intensity raw value close to background levels. Probe sets for each array are pre-processed using RMA. Also, probe sets whose expression change under all experimental conditions was below a threshold, based on the standard desviation of the normalised intensity values, were filtered.

Dataset Information

Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection.

Publications

Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection.

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