scholarly journals Stimulating Respiratory Activity Primes Anaerobically Grown Listeria monocytogenes for Subsequent Intracellular Infections

Pathogens ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 96 ◽  
Author(s):  
Nathan Wallace ◽  
Erica Rinehart ◽  
Yvonne Sun
Keyword(s):  
Listeria Monocytogenes ◽  
Respiratory Activity ◽  
Growth Conditions ◽  
Growth Defect ◽  
Listeriolysin O ◽  
Low Oxygen ◽  
Intracellular Growth ◽  
Aerobic Conditions ◽  
Intracellular Infections ◽  
Cell Spread

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, enteric pathogen and the causative agent of listeriosis. During transition through the gastrointestinal tract, L. monocytogenes routinely encounters suboxic conditions. However, how the exposure to the low oxygen environment affects subsequent pathogenesis is not completely understood. Our lab previously reported that anaerobically grown L. monocytogenes exhibited an intracellular growth defect in macrophages even though the infection took place under aerobic conditions. This phenotype suggests that prior growth conditions have a prolonged effect on the outcome of subsequent intracellular infection. In this study, to further investigate the mechanisms that contribute to the compromised intracellular growth after anaerobic exposure, we hypothesized that the lack of respiratory activity under anaerobic conditions prevented anaerobically grown L. monocytogenes to establish subsequent intracellular growth under aerobic conditions. To test this hypothesis, respiratory activity in anaerobically grown L. monocytogenes was stimulated by exogenous fumarate and subsequent intracellular pathogenesis was assessed. The results showed that fumarate supplementation significantly increased the respiratory activity of anaerobically grown L. monocytogenes and rescued the subsequent intracellular growth defect, likely through promoting the production of listeriolysin O, phagosomal escape, and cell-cell spread. This study highlights the importance of respiratory activity in L. monocytogenes in modulating the outcome of subsequent intracellular infections.

scholarly journals Inducible Control of Virulence Gene Expression in Listeria monocytogenes: Temporal Requirement of Listeriolysin O during Intracellular Infection

2002 ◽  
Vol 184 (21) ◽  
pp. 5935-5945 ◽  
Author(s):  
Christina E. Dancz ◽  
Andrea Haraga ◽  
Daniel A. Portnoy ◽  
Darren E. Higgins
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Line ◽  
Broth Culture ◽  
Listeriolysin O ◽  
Virulence Determinants ◽  
Intracellular Growth ◽  
Iptg Induction ◽  
Intracellular Infection ◽  
Dose Dependent ◽  
Cell Spread

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.

scholarly journals Characterization of Listeria monocytogenes Expressing Anthrolysin O and Phosphatidylinositol-Specific Phospholipase C from Bacillus anthracis

2005 ◽  
Vol 73 (10) ◽  
pp. 6639-6646 ◽  
Author(s):  
Zhengyu Wei ◽  
Pamela Schnupf ◽  
Mathilde A. Poussin ◽  
Lauren A. Zenewicz ◽  
Hao Shen ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Bacillus Anthracis ◽  
Host Cell ◽  
Phospholipase C ◽  
Host Cells ◽  
Listeriolysin O ◽  
Intracellular Growth ◽  
Cell Cytosol ◽  
Anthrolysin O ◽  
Cell Spread

ABSTRACT Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.

scholarly journals EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

2020 ◽  
Author(s):  
Jeanine Rismondo ◽  
Lisa M. Schulz ◽  
Maria Yacoub ◽  
Ashima Wadhawan ◽  
Michael Hoppert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Abc Transporter ◽  
Cell Lysis ◽  
Growth Conditions ◽  
Cell Wall Integrity ◽  
Growth Defect ◽  
Cell Wall Biosynthesis ◽  
High Concentrations

Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity. IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

scholarly journals Role of Listeriolysin O in Cell-to-Cell Spread ofListeria monocytogenes

2000 ◽  
Vol 68 (2) ◽  
pp. 999-1003 ◽  
Author(s):  
Margaret M. Gedde ◽  
Darren E. Higgins ◽  
Lewis G. Tilney ◽  
Daniel A. Portnoy
Keyword(s):  
Bacterial Pathogen ◽  
Listeriolysin O ◽  
Genetic Approach ◽  
Wild Type ◽  
Intracellular Growth ◽  
Double Membrane ◽  
Vacuolar Compartment ◽  
Oflisteria Monocytogenes ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape ofL. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

scholarly journals The Production of Listeriolysin O and Subsequent Intracellular Infections by Listeria monocytogenes Are Regulated by Exogenous Short Chain Fatty Acid Mixtures

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 218
Author(s):  
Erica Rinehart ◽  
Julia Chapman ◽  
Yvonne Sun
Keyword(s):  
Listeria Monocytogenes ◽  
Foodborne Pathogen ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Listeriolysin O ◽  
Subsequent Infection ◽  
Low Levels ◽  
Isogenic Mutants ◽  
Intracellular Infections ◽  
Fatty Acid Mixtures

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

scholarly journals Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

2003 ◽  
Vol 185 (21) ◽  
pp. 6295-6307 ◽  
Author(s):  
Angelika Gründling ◽  
Mark D. Gonzalez ◽  
Darren E. Higgins
Keyword(s):  
Listeria Monocytogenes ◽  
Epithelial Cells ◽  
Host Cell ◽  
Phospholipase C ◽  
Expression System ◽  
Broth Culture ◽  
Listeriolysin O ◽  
Proteolytic Activation ◽  
Human Epithelial Cells ◽  
Cell Spread

ABSTRACT In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

scholarly journals EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

2020 ◽  
Author(s):  
Jeanine Rismondo ◽  
Lisa M. Schulz ◽  
Maria Yacoub ◽  
Ashima Wadhawan ◽  
Michael Hoppert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Abc Transporter ◽  
Cell Lysis ◽  
Growth Conditions ◽  
Cell Wall Integrity ◽  
Growth Defect ◽  
Cell Wall Biosynthesis ◽  
High Concentrations

ABSTRACTLysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCEThe ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

scholarly journals The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis

2009 ◽  
Vol 77 (7) ◽  
pp. 2612-2623 ◽  
Author(s):  
Francis Alonzo ◽  
Gary C. Port ◽  
Min Cao ◽  
Nancy E. Freitag
Keyword(s):  
Listeria Monocytogenes ◽  
Gene Product ◽  
Bacterial Pathogen ◽  
Virulence Gene ◽  
Host Cells ◽  
Listeriolysin O ◽  
Virulence Gene Expression ◽  
High Concentrations ◽  
Host Infection ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection.

scholarly journals Salmonella enterica Serovar Gallinarum Requires ppGpp for Internalization and Survival in Animal Cells

2008 ◽  
Vol 190 (19) ◽  
pp. 6340-6350 ◽  
Author(s):  
Jae-Ho Jeong ◽  
Miryoung Song ◽  
Sang-Ik Park ◽  
Kyoung-Oh Cho ◽  
Joon Haeng Rhee ◽  
...  
Keyword(s):  
Growth Phase ◽  
Salmonella Enterica ◽  
Animal Cell ◽  
Growth Conditions ◽  
Exponential Growth Phase ◽  
Low Oxygen ◽  
Intracellular Growth ◽  
Animal Cells ◽  
Cell Internalization ◽  
Serovar Typhimurium

ABSTRACT To elucidate the pathogenic mechanism of Salmonella enterica serovar Gallinarum, we examined the expression of the genes encoded primarily in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. These genes were found to be induced as cultures entered stationary phase under high- and low-oxygen growth conditions, as also observed for Salmonella serovar Typhimurium. In contrast, Salmonella serovar Gallinarum in the exponential growth phase most efficiently internalized cultured animal cells. Analysis of mutants defective in SPI-1 genes, SPI-2 genes, and others implicated in early stages of infection revealed that SPI-1 genes were not involved in the internalization of animal cells by Salmonella serovar Gallinarum. Following entry, however, Salmonella serovar Gallinarum was found to reside in LAMP1-positive vacuoles in both phagocytic and nonphagocytic cells, although internalization was independent of SPI-1. A mutation that conferred defects in ppGpp synthesis was the only one found to affect animal cell internalization by Salmonella serovar Gallinarum. It was concluded that Salmonella serovar Gallinarum internalizes animal cells by a mechanism independent of SPI-1 genes but dependent on ppGpp. Intracellular growth also required ppGpp for the transcription of genes encoded in SPI-2.

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