Deletion of glutaredoxin promotes oxidative tolerance and intracellular infection in Listeria monocytogenes

ABSTRACT We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes. An L. monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent promoter. Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L. monocytogenes-containing phagosomes. Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture. Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG. We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth. Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line. Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent. The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed.

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pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes

Molecular Microbiology ◽

10.1046/j.1365-2958.2000.01708.x ◽

2000 ◽

Vol 35 (2) ◽

pp. 289-298 ◽

Cited By ~ 63

Author(s):

Helene Marquis ◽

Elizabeth J. Hager

Keyword(s):

Listeria Monocytogenes ◽

Phospholipase C ◽

Intracellular Infection

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Comparative activity of quinolones (ciprofloxacin, levofloxacin, moxifloxacin and garenoxacin) against extracellular and intracellular infection by Listeria monocytogenes and Staphylococcus aureus in J774 macrophages

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dki059 ◽

2005 ◽

Vol 55 (4) ◽

pp. 511-517 ◽

Cited By ~ 42

Author(s):

C. Seral ◽

M. Barcia-Macay ◽

M. P. Mingeot-Leclercq ◽

P. M. Tulkens ◽

F. Van Bambeke

Keyword(s):

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Intracellular Infection ◽

And Staphylococcus Aureus ◽

Comparative Activity

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The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection

Journal of Bacteriology ◽

10.1128/jb.01168-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3594-3603 ◽

Cited By ~ 10

Author(s):

Heather S. O'Neil ◽

Brian M. Forster ◽

Kari L. Roberts ◽

Andrew J. Chambers ◽

Alan Pavinski Bitar ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Catalytic Domain ◽

Bacterial Pathogen ◽

Folding Kinetics ◽

Wild Type ◽

Semisolid Medium ◽

Mutant Proteins ◽

Intracellular Infection ◽

Infected Cells

ABSTRACT Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (MplΔpro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mplΔpro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.

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An M29 Aminopeptidase from Listeria Monocytogenes Contributes to In Vitro Bacterial Growth but not to Intracellular Infection

Microorganisms ◽

10.3390/microorganisms8010110 ◽

2020 ◽

Vol 8 (1) ◽

pp. 110 ◽

Cited By ~ 1

Author(s):

Xian Zhang ◽

Chiyu Guan ◽

Yi Hang ◽

Fengdan Liu ◽

Jing Sun ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Bacterial Growth ◽

Optimal Growth ◽

Defined Medium ◽

Intracellular Infection ◽

Physiological Processes ◽

Wide Range ◽

Catalytic Motif ◽

Listeria Infection

Aminopeptidases that catalyze the removal of N-terminal residues from polypeptides or proteins are crucial for physiological processes. Here, we explore the biological functions of an M29 family aminopeptidase II from Listeria monocytogenes (LmAmpII). We show that LmAmpII contains a conserved catalytic motif (EEHYHD) that is essential for its enzymatic activity and LmAmpII has a substrate preference for arginine and leucine. Studies on biological roles indicate that LmAmpII is required for in vitro growth in a chemically defined medium for optimal growth of L. monocytogenes but is not required for bacterial intracellular infection in epithelial cells and macrophages, as well as cell-to-cell spreading in fibroblasts. Moreover, LmAmpII is found as dispensable for bacterial pathogenicity in mice. Taken together, we conclude that LmAmpII, an M29 family aminopeptidase, can efficiently hydrolyze a wide range of substrates and is required for in vitro bacterial growth, which lays a foundation for in-depth investigations of aminopeptidases as potential targets to defend Listeria infection.

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