scholarly journals Cardiotropic isolates of Listeria monocytogenes with enhanced vertical transmission dependent upon the bacterial surface protein InlB

2020 ◽  
Author(s):  
Nicole M. Lamond ◽  
P. David McMullen ◽  
Dhanendra Paramasvaran ◽  
Lavanya Visvahabrathy ◽  
Samuel J. Eallanardo ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Vertical Transmission ◽  
Cardiac Tissue ◽  
Genetic Manipulation ◽  
Surface Protein ◽  
Placental Tissue ◽  
Human Trophoblast ◽  
Bacterial Surface ◽  
Culture Cells ◽  
Fetal Infection

Listeria monocytogenes is a facultative gram-positive intracellular bacterium that is capable of causing serious invasive infections in pregnant women resulting in abortion, still-birth, and disseminated fetal infection. Previously, a clinical L. monocytogenes isolate, 07PF0776, was identified as having an enhanced ability to target cardiac tissue; this tissue tropism appeared to correlate with amino acid variations found within Internalin B (InlB), a bacterial surface protein associated with host cell invasion. Given that the mammalian receptor bound by InlB, Met, is abundantly expressed by placental tissue, we assessed 07PF0776 for its ability to be transmitted from mother to fetus. Pregnant Swiss-Webster mice were infected on gestational day E13 via tail vein injection with the standard isolate 10403S, a non-cardiotropic strain, or 07PF0776, the cardiac isolate. Pregnant mice infected with 07PF0776 exhibited significantly enhanced transmission of L. monocytogenes to placentas and fetuses compared to 10403S. Both bacterial burdens and the frequency of placental and fetal infection were increased in mice infected with the cardiac isolate. Strain 07PF0776 also exhibited an enhanced ability to invade Jar human trophoblast tissue culture cells in comparison to 10403S, and was found to have increased levels of InlB associated with the bacterial cell surface. Overexpression of surface InlB via genetic manipulation was sufficient to confer enhanced invasion of the placenta and fetus to both 10403S and 07PF0776. These data support a central role for surface InlB in promoting vertical transmission of L. monocytogenes.

scholarly journals Vertical Transmission of Listeria monocytogenes: Probing the Balance between Protection from Pathogens and Fetal Tolerance

Pathogens ◽  
2018 ◽  
Vol 7 (2) ◽  
pp. 52 ◽  
Author(s):  
Nicole Lamond ◽  
Nancy Freitag
Keyword(s):  
Cell Culture ◽  
Listeria Monocytogenes ◽  
Animal Models ◽  
Vertical Transmission ◽  
Surface Proteins ◽  
Placental Barrier ◽  
Bacterial Surface ◽  
Organ Models ◽  
The Many

Protection of the developing fetus from pathogens is one of the many critical roles of the placenta. Listeria monocytogenes is one of a select number of pathogens that can cross the placental barrier and cause significant harm to the fetus, leading to spontaneous abortion, stillbirth, preterm labor, and disseminated neonate infection despite antibiotic treatment. Such severe outcomes serve to highlight the importance of understanding how L. monocytogenes mediates infiltration of the placental barrier. Here, we review what is currently known regarding vertical transmission of L. monocytogenes as a result of cell culture and animal models of infection. In vitro cell culture and organ models have been useful for the identification of L. monocytogenes virulence factors that contribute to placental invasion. Examples include members of the Internalin family of bacterial surface proteins such as Interalin (Inl)A, InlB, and InlP that promote invasion of cells at the maternal-fetal interface. A number of animal models have been used to interrogate L. monocytogenes vertical transmission, including mice, guinea pigs, gerbils, and non-human primates; each of these models has advantages while still not providing a comprehensive understanding of L. monocytogenes invasion of the human placenta and/or fetus. These models do, however, allow for the molecular investigation of the balance between fetal tolerance and immune protection from L. monocytogenes during pregnancy.

Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly

1993 ◽  
Vol 105 (3) ◽  
pp. 699-710 ◽  
Author(s):  
C. Kocks ◽  
R. Hellio ◽  
P. Gounon ◽  
H. Ohayon ◽  
P. Cossart
Keyword(s):  
Listeria Monocytogenes ◽  
Surface Protein ◽  
Intracellular Bacteria ◽  
Intracellular Pathogen ◽  
Actin Assembly ◽  
Bacterial Gene ◽  
Bacterial Surface ◽  
Left Behind ◽  
Bacterial Division ◽  
Host Tissues

The facultative intracellular pathogen Listeria monocytogenes can infect host tissues by using directional actin assembly to propel itself from one cell into another. The movement is generated by continuous actin assembly from one end of the bacterium into a tail, which is left behind in the cytoplasm. Bacterial actin assembly requires expression of the bacterial gene actA. We have used immunocytochemistry to show that the actA gene product, ActA, is distributed asymmetrically on the bacterial surface: it is not expressed at one pole and is increasingly concentrated towards the other. This polarized distribution of ActA was linked to bacterial division: ActA protein was not, or only faintly, expressed at the pole that had been formed during the previous division. On intracellular bacteria ActA was expressed at the site of actin assembly, suggesting that ActA may be involved in actin filament nucleation off the bacterial surface. We predict that the asymmetrical distribution of this protein is required for the ability of intracellular Listeria to move in the direction of the non-ActA expressing pole.

scholarly journals Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex

2000 ◽  
Vol 113 (18) ◽  
pp. 3277-3287 ◽  
Author(s):  
S. Pistor ◽  
L. Grobe ◽  
A.S. Sechi ◽  
E. Domann ◽  
B. Gerstel ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Filament ◽  
Mutational Analysis ◽  
Surface Protein ◽  
Specific Interactions ◽  
Latrunculin B ◽  
Bacterial Surface ◽  
Arginine Residues ◽  
Infected Cells ◽  
Intracellular Motility

The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.

scholarly journals The Host Scaffolding Protein Filamin A and the Exocyst Complex Control Exocytosis during InlB-Mediated Entry of Listeria monocytogenes

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
Manmeet Bhalla ◽  
Hoan Van Ngo ◽  
Gaurav Chandra Gyanwali ◽  
Keith Ireton
Keyword(s):  
Listeria Monocytogenes ◽  
Surface Protein ◽  
Scaffolding Protein ◽  
Signaling Proteins ◽  
Filamin A ◽  
Content Type ◽  
Bacterial Surface ◽  
Kinase C ◽  
Exocyst Complex ◽  
Mtor Protein

ABSTRACT Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry, in part, through stimulation of localized exocytosis. How exocytosis is upregulated during entry is not understood. Here, we show that the human signaling proteins mTOR, protein kinase C-α (PKC-α), and RalA promote exocytosis during entry by controlling the scaffolding protein Filamin A (FlnA). InlB-mediated uptake was accompanied by PKC-α–dependent phosphorylation of serine 2152 in FlnA. Depletion of FlnA by RNA interference (RNAi) or expression of a mutated FlnA protein defective in phosphorylation impaired InlB-dependent internalization. These findings indicate that phosphorylation of FlnA by PKC-α contributes to entry. mTOR and RalA were found to mediate the recruitment of FlnA to sites of InlB-mediated entry. Depletion of PKC-α, mTOR, or FlnA each reduced exocytosis during InlB-mediated uptake. Because the exocyst complex is known to mediate polarized exocytosis, we examined if PKC-α, mTOR, RalA, or FlnA affects this complex. Depletion of PKC-α, mTOR, RalA, or FlnA impaired recruitment of the exocyst component Exo70 to sites of InlB-mediated entry. Experiments involving knockdown of Exo70 or other exocyst proteins demonstrated an important role for the exocyst complex in uptake of Listeria. Collectively, our results indicate that PKC-α, mTOR, RalA, and FlnA comprise a signaling pathway that mobilizes the exocyst complex to promote infection by Listeria.

scholarly journals The Host GTPase Arf1 and Its Effectors AP1 and PICK1 Stimulate Actin Polymerization and Exocytosis To Promote Entry of Listeria monocytogenes

2019 ◽  
Vol 88 (2) ◽  
Author(s):  
Susan Saila ◽  
Gaurav Chandra Gyanwali ◽  
Mazhar Hussain ◽  
Antonella Gianfelice ◽  
Keith Ireton
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Surface Protein ◽  
Dominant Negative ◽  
Host Cells ◽  
Filamin A ◽  
Content Type ◽  
Bacterial Surface ◽  
Dominant Negative Allele

ABSTRACT Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.

scholarly journals Critical Role for the Host GTPase-Activating Protein ARAP2 in InlB-Mediated Entry of Listeria monocytogenes

2010 ◽  
Vol 78 (11) ◽  
pp. 4532-4541 ◽  
Author(s):  
Balramakrishna Gavicherla ◽  
Lisa Ritchey ◽  
Antonella Gianfelice ◽  
Andrey A. Kolokoltsov ◽  
Robert A. Davey ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Critical Role ◽  
Bacterial Pathogen ◽  
Surface Protein ◽  
Rho Proteins ◽  
Gtpase Activating Protein ◽  
Bacterial Surface ◽  
Downstream Signaling ◽  
Binding Domains

ABSTRACT The bacterial pathogen Listeria monocytogenes causes food-borne illnesses culminating in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. Interaction of InlB with the Met receptor elicits host downstream signaling pathways that promote F-actin cytoskeletal changes responsible for pathogen engulfment. Here we show that the mammalian signaling protein ARAP2 plays a critical role in cytoskeletal remodeling and internalization of Listeria. Depletion of ARAP2 through RNA interference (RNAi) caused a marked inhibition of InlB-mediated F-actin rearrangements and bacterial entry. ARAP2 contains multiple functional domains, including a GTPase-activating protein (GAP) domain that antagonizes the GTPase Arf6 and a domain capable of binding the GTPase RhoA. Genetic data indicated roles for both the Arf GAP and RhoA binding domains in Listeria entry. Experiments involving Arf6 RNAi or a constitutively activated allele of Arf6 demonstrated that one of the ways in which ARAP2 promotes bacterial uptake is by restraining the activity of Arf6. Conversely, Rho activity was dispensable for Listeria internalization, suggesting that the RhoA binding domain in ARAP2 acts by engaging a host ligand other than Rho proteins. Collectively, our findings indicate that ARAP2 promotes InlB-mediated entry of Listeria, in part, by antagonizing the host GTPase Arf6.

scholarly journals The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.

1996 ◽  
Vol 135 (3) ◽  
pp. 647-660 ◽  
Author(s):  
G A Smith ◽  
J A Theriot ◽  
D A Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Actin Polymerization ◽  
Consensus Sequence ◽  
Wild Type ◽  
Movement Rate ◽  
Bacterial Surface ◽  
Rate Dependent ◽  
Repeat Domain ◽  
Low Levels ◽  
Rate Of Movement

The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

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