scholarly journals Actin Polymerization Drives Septation ofListeria monocytogenes namAHydrolase Mutants, Demonstrating Host Correction of a Bacterial Defect

2011 ◽  
Vol 79 (4) ◽  
pp. 1458-1470 ◽  
Author(s):  
Francis Alonzo ◽  
P. David McMullen ◽  
Nancy E. Freitag
Keyword(s):  
Cell Wall ◽  
Host Cell ◽  
Exponential Growth ◽  
Bacterial Cell ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Broth Culture ◽  
Gram Positive ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACTThe Gram-positive bacterial cell wall presents a structural barrier that requires modification for protein secretion and large-molecule transport as well as for bacterial growth and cell division. The Gram-positive bacteriumListeria monocytogenesadjusts cell wall architecture to promote its survival in diverse environments that include soil and the cytosol of mammalian cells. Here we provide evidence for the enzymatic flexibility of the murein hydrolase NamA and demonstrate that bacterial septation defects associated with a loss of NamA are functionally complemented by physical forces associated with actin polymerization within the host cell cytosol.L. monocytogenesΔnamAmutants formed long bacterial chains during exponential growth in broth culture; however, normal septation could be restored if mutant cells were cocultured with wild-typeL. monocytogenesbacteria or by the addition of exogenous NamA. Surprisingly, ΔnamAmutants were not significantly attenuated for virulence in mice despite the pronounced exponential growth septation defect. The physical force ofL. monocytogenes-mediated actin polymerization within the cytosol was sufficient to sever ΔnamAmutant intracellular chains and thereby enable the process of bacterial cell-to-cell spread so critical forL. monocytogenesvirulence. The inhibition of actin polymerization by cytochalasin D resulted in extended intracellular bacterial chains for which septation was restored following drug removal. Thus, despite the requirement for NamA for the normal septation of exponentially growingL. monocytogenescells, the hydrolase is essentially dispensable onceL. monocytogenesgains access to the host cell cytosol. This phenomenon represents a notable example of eukaryotic host cell complementation of a bacterial defect.

scholarly journals Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis

2016 ◽  
Vol 84 (5) ◽  
pp. 1603-1614 ◽  
Author(s):  
Carina Carraro Pessoa ◽  
Éden Ramalho Ferreira ◽  
Ethel Bayer-Santos ◽  
Michel Rabinovitch ◽  
Renato Arruda Mortara ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Leishmania Amazonensis ◽  
Content Type ◽  
Multidimensional Imaging ◽  
Protozoan Infections ◽  
Cell Cytosol ◽  
Host Cell Cytosol

The trypanosomatidsLeishmania amazonensisandTrypanosoma cruziare excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellatesL. amazonensisandT. cruzilodge within acidified parasitophorous vacuoles (PVs). However, whereasL. amazonensisdevelops in spacious, phagolysosome-like PVs that may enclose numerous parasites,T. cruziis transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation ofT. cruzi, we constructed chimeric vacuoles by infection ofL. amazonensisamastigote-infected macrophages withT. cruziepimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate ofT. cruziinL. amazonensisPVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that althoughT. cruziEPIs remained motile and conserved their morphology in chimeric vacuoles,T. cruziMTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles ofL. amazonensisare permissive toT. cruzisurvival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude thatT. cruzidifferentiation can take place inLeishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.

scholarly journals Restricted Translocation across the Cell Wall Regulates Secretion of the Broad-Range Phospholipase C of Listeria monocytogenes

2003 ◽  
Vol 185 (20) ◽  
pp. 5953-5958 ◽  
Author(s):  
Aleksandra Snyder ◽  
Hélène Marquis
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Bacterial Cell ◽  
Active Form ◽  
Bacterial Cell Wall ◽  
Proteolytic Activation ◽  
Cell Cytosol ◽  
Rate Limiting ◽  
Host Cell Cytosol

ABSTRACT The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain. Mpl, a secreted metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH. pH-regulated release of active PC-PLC is Mpl dependent. To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated. Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight. Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall. Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol. These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles.

scholarly journals Listeria monocytogenes PrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol

2010 ◽  
Vol 78 (11) ◽  
pp. 4944-4957 ◽  
Author(s):  
Francis Alonzo ◽  
Nancy E. Freitag
Keyword(s):  
Listeria Monocytogenes ◽  
Host Cell ◽  
Host Cells ◽  
Broth Culture ◽  
Cell Infection ◽  
Bacterial Viability ◽  
Cell Cytosol ◽  
Critical Requirement ◽  
Host Cell Cytosol

ABSTRACT In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection.

scholarly journals Telavancin, a Multifunctional Lipoglycopeptide, Disrupts both Cell Wall Synthesis and Cell Membrane Integrity in Methicillin-Resistant Staphylococcus aureus

2005 ◽  
Vol 49 (3) ◽  
pp. 1127-1134 ◽  
Author(s):  
Deborah L. Higgins ◽  
Ray Chang ◽  
Dmitri V. Debabov ◽  
Joey Leung ◽  
Terry Wu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Bactericidal Activity ◽  
Bacterial Cell ◽  
Cell Membrane Potential ◽  
Gram Positive ◽  
Methicillin Resistant ◽  
Bacterial Cell Membrane

ABSTRACTThe emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptideN,N′-diacetyl-l-lysinyl-d-alanyl-d-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistantStaphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K+. The timing of these changes correlated with rapid , concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.

Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes

1999 ◽  
Vol 112 (5) ◽  
pp. 681-693
Author(s):  
U.E. Schaible ◽  
P.H. Schlesinger ◽  
T.H. Steinberg ◽  
W.F. Mangel ◽  
T. Kobayashi ◽  
...  
Keyword(s):  
Host Cell ◽  
Lucifer Yellow ◽  
Cysteine Proteinase ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Anion Transporter ◽  
Infected Cells ◽  
Lysobisphosphatidic Acid ◽  
Cell Cytosol ◽  
Host Cell Cytosol

The intracellular parasite Leishmania survives and proliferates in host macrophages. In this study we show that parasitophorous vacuoles of L. mexicana gain access to cytosolic material via two different routes. (1) Small anionic molecules such as Lucifer Yellow are rapidly transported into the vacuoles by an active transport mechanism that is sensitive to inhibitors of the host cell's organic anion transporter. (2) Larger molecules such as fluorescent dextrans introduced into the host cell cytosol are also delivered to parasitophorous vacuoles. This transport is slower and sensitive to modulators of autophagy. Infected macrophages were examined by two novel assays to visualize and quantify this process. Immunoelectron microscopy of cells loaded with digoxigenin-dextran revealed label in multivesicular endosomes, which appeared to fuse with parasitophorous vacuoles. The inner membranes of the multivesicular vesicles label strongly with antibodies against lysobisphosphatidic acid, suggesting that they represent a point of confluence between the endosomal and autophagosomal pathways. Although the rate of autophagous transfer was comparable in infected and uninfected cells, infected cells retained hydrolyzed cysteine proteinase substrate to a greater degree. These data suggest that L. mexicana-containing vacuoles have access to potential nutrients in the host cell cytosol via at least two independent mechanisms.

scholarly journals Type II Secretion Substrates ofLegionella pneumophilaTranslocate Out of the Pathogen-Occupied Vacuole via a Semipermeable Membrane

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Hilary K. Truchan ◽  
Harry D. Christman ◽  
Richard C. White ◽  
Nakisha S. Rutledge ◽  
Nicholas P. Cianciotto
Keyword(s):  
Host Cell ◽  
Infected Host ◽  
Type Ii Secretion ◽  
Dependent Manner ◽  
Type Ii ◽  
Murine Macrophages ◽  
Intracellular Infection ◽  
Infected Host Cell ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACTLegionella pneumophilareplicates in macrophages in a host-derived phagosome, termed theLegionella-containing vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into the macrophage cytosol is well established, the location of type II secretion (T2S) substrates in the infected host cell is unknown. Here, we show that the T2S substrate ProA, a metalloprotease, translocates into the cytosol of human macrophages, where it associates with the LCV membrane (LCVM). Translocation is detected as early as 10 h postinoculation (p.i.), which is approximately the midpoint of the intracellular life cycle. However, it is detected as early as 6 h p.i. if ProA is hyperexpressed, indicating that translocation depends on the timing of ProA expression and that any other factors necessary for translocation are in place by that time point. Translocation occurs with allL. pneumophilastrains tested and in amoebae, natural hosts forL. pneumophila. It was absent in murine bone marrow-derived macrophages and murine macrophage cell lines. The ChiA chitinase also associated with the cytoplasmic face of the LCVM at 6 h p.i. and in a T2S-dependent manner. Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced by damage to host membranes, appeared within the LCV of infected human but not murine macrophages beginning at 6 h p.i. Thus, we hypothesize that ProA and ChiA are first secreted into the vacuolar lumen by the activity of the T2S and subsequently traffic into the macrophage cytosol via a novel mechanism that involves a semipermeable LCVM.IMPORTANCEInfection of macrophages and amoebae plays a central role in the pathogenesis ofL. pneumophila, the agent of Legionnaires’ disease. We have previously demonstrated that the T2S system ofL. pneumophilagreatly contributes to intracellular infection. However, the location of T2S substrates within the infected host cell is unknown. This report presents the first evidence of aL. pneumophilaT2S substrate in the host cell cytosol and, therefore, the first evidence of a non-T4S effector trafficking out of the LCV. We also provide the first indication that the LCV is not completely intact but is instead semipermeable and that this occurs in human but not murine macrophages. Given this permeability, we hypothesize that other T2S substrates and LCV lumenal contents can escape into the host cell cytosol. Thus, these substrates may represent a battery of previously unidentified effectors that can interact with host factors and contribute to intracellular infection byL. pneumophila.

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