scholarly journals Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

2015 ◽  
Vol 83 (8) ◽  
pp. 3233-3242 ◽  
Author(s):  
Lena Meyer ◽  
Jeanette E. Bröms ◽  
Xijia Liu ◽  
Martin E. Rottenberg ◽  
Anders Sjöstedt
Keyword(s):  
Listeria Monocytogenes ◽  
Francisella Tularensis ◽  
Hela Cells ◽  
Cell Types ◽  
Metabolic Adaptation ◽  
Content Type ◽  
Bacterial Uptake ◽  
J774 Cells ◽  
Cell Cytosol ◽  
Successful Replication

Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. ForFrancisella tularensis, many genes of theFrancisellapathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) ofF. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpEstrains; andListeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC−/−BMDM, MyD88−/−BMDM, J774 cells, or HeLa cells, LVS, ΔpdpEand ΔiglGmutants, andL. monocytogenesreplicated efficiently in all five cell types, whereas the ΔiglAand ΔiglCmutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC−/−BMDM, and HeLa cells. In contrast to the rapid replication in other cell types,L. monocytogenesshowed no replication in MyD88−/−BMDM and LVS showed no replication in either BMDM or MyD88−/−BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartmentper seare important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

scholarly journals Recent advances in understanding Listeria monocytogenes infection: the importance of subcellular and physiological context

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1126 ◽  
Author(s):  
Daryl J. V. David ◽  
Pascale Cossart
Keyword(s):  
Listeria Monocytogenes ◽  
Gut Microbiota ◽  
Bacterial Pathogen ◽  
Cell Types ◽  
Infection Process ◽  
Foodborne Disease ◽  
Listeria Monocytogenes Infection ◽  
Cell Cytosol ◽  
Critical Components ◽  
Host Cell Cytosol

The bacterial pathogen Listeria monocytogenes (Lm) is the causative agent of listeriosis, a rare but fatal foodborne disease. During infection, Lm can traverse several host barriers and enter the cytosol of a variety of cell types. Thus, consideration of the extracellular and intracellular niches of Lm is critical for understanding the infection process. Here, we review advances in our understanding of Lm infection and highlight how the interactions between the host and the pathogen are context dependent. We discuss discoveries of how Lm senses entry into the host cell cytosol. We present findings concerning how the nature of the various cytoskeleton components subverted by Lm changes depending on both the stage of infection and the subcellular context. We present discoveries of critical components required for Lm traversal of physiological barriers. Interactions between the host gut microbiota and Lm will be briefly discussed. Finally, the importance of Lm biodiversity and post-genomics approaches as a promising way to discover novel virulence factors will be highlighted.

scholarly journals A Redox-Responsive Transcription Factor Is Critical for Pathogenesis and Aerobic Growth of Listeria monocytogenes

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Aaron T. Whiteley ◽  
Brittany R. Ruhland ◽  
Mauna B. Edrozo ◽  
Michelle L. Reniere
Keyword(s):  
Oxidative Stress ◽  
Listeria Monocytogenes ◽  
Aerobic Growth ◽  
Redox Stress ◽  
Content Type ◽  
Redox Responsive ◽  
Cell Cytosol ◽  
Redox Sensing ◽  
Anaerobic Environment

ABSTRACT Bacterial pathogens have evolved sophisticated mechanisms to sense and adapt to redox stress in nature and within the host. However, deciphering the redox environment encountered by intracellular pathogens in the mammalian cytosol is challenging, and that environment remains poorly understood. In this study, we assessed the contributions of the two redox-responsive, Spx-family transcriptional regulators to the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular pathogen. Spx-family proteins are highly conserved in Firmicutes, and the L. monocytogenes genome contains two paralogues, spxA1 and spxA2. Here, we demonstrate that spxA1, but not spxA2, is required for the oxidative stress response and pathogenesis. SpxA1 function appeared to be conserved with the Bacillus subtilis homologue, and resistance to oxidative stress required the canonical CXXC redox-sensing motif. Remarkably, spxA1 was essential for aerobic growth, demonstrating that L. monocytogenes SpxA1 likely regulates a distinct set of genes. Although the ΔspxA1 mutant did not grow in the presence of oxygen in the laboratory, it was able to replicate in macrophages and colonize the spleens, but not the livers, of infected mice. These data suggest that the redox state of bacteria during infection differs significantly from that of bacteria growing in vitro. Further, the host cell cytosol may resemble an anaerobic environment, with tissue-specific variations in redox stress and oxygen concentration.

scholarly journals Secretion Chaperones PrsA2 and HtrA Are Required for Listeria monocytogenes Replication following Intracellular Induction of Virulence Factor Secretion

2016 ◽  
Vol 84 (10) ◽  
pp. 3034-3046 ◽  
Author(s):  
Jana K. Ahmed ◽  
Nancy E. Freitag
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Factor ◽  
Bacterial Growth ◽  
Cell Types ◽  
Broth Culture ◽  
Bacterial Invasion ◽  
Bacterial Membrane ◽  
Content Type ◽  
Factor Secretion

The Gram-positive bacteriumListeria monocytogenestransitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane.L. monocytogenesstrains that lack bothprsA2andhtrAexhibit near-normal patterns of growth in broth culture but are severely attenuatedin vivo. We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele ofprfA(prfA*) into ΔprsA2ΔhtrAstrains was found to essentially inhibit bacterial growth at 37°C in broth culture. ΔprsA2ΔhtrAstrains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion.

scholarly journals Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes

2015 ◽  
Vol 83 (9) ◽  
pp. 3740-3748 ◽  
Author(s):  
Jiahui Wang ◽  
Jane E. King ◽  
Marie Goldrick ◽  
Martin Lowe ◽  
Frank B. Gertler ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Host Cell ◽  
Foodborne Pathogen ◽  
Cell Types ◽  
Ph Domain ◽  
Content Type ◽  
Bacterial Surface ◽  
Binding Domains ◽  
Interfering Rna ◽  
Cell Spread

Listeria monocytogenesis a foodborne pathogen capable of invading a broad range of cell types and replicating within the host cell cytoplasm. This paper describes the colocalization of host cell lamellipodin (Lpd) with intracellularL. monocytogenesdetectable 6 h postinfection of epithelial cells. The association was mediated via interactions between both the peckstrin homology (PH) domain in Lpd and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] on the bacterial surface and by interactions between the C-terminal EVH1 (Ena/VASP [vasodilator-stimulated phosphoprotein] homology domain 1) binding domains of Lpd and the host VASP (vasodilator-stimulated phosphoprotein) recruited to the bacterial cell surface by the listerial ActA protein. Depletion of Lpd by short interfering RNA (siRNA) resulted in reduced plaque size and number, indicating a role for Lpd in cell-to-cell spread. In contrast, overexpression of Lpd resulted in an increase in the number ofL. monocytogenes-containing protrusions (listeriopods). Manipulation of the levels of Lpd within the cell also affected the intracellular velocity ofL. monocytogenes, with a reduction in Lpd corresponding to an increase in intracellular velocity. These data, together with the observation that Lpd accumulated at the interface between the bacteria and the developing actin tail at the initiation of actin-based movement, indicate a possible role for Lpd in the actin-based movement and the cell-to-cell spread ofL. monocytogenes.

scholarly journals A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Grischa Y. Chen ◽  
Courtney E. McDougal ◽  
Marc A. D’Antonio ◽  
Jonathan L. Portman ◽  
John-Demian Sauer
Keyword(s):  
Listeria Monocytogenes ◽  
Francisella Tularensis ◽  
Humoral Immunity ◽  
Bacterial Colonization ◽  
Bacterial Pathogens ◽  
Cell Types ◽  
Genetic Screen ◽  
Cellular Respiration ◽  
Nutritional Immunity ◽  
Transport Chain

ABSTRACT Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity. IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis, are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role for the synthesis of the menaquinone precursor 1,4-dihydroxy-2-naphthoate (DHNA) in cytosolic survival. Together, these data begin to elucidate adaptations that allow cytosolic pathogens to survive in their intracellular niches. IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis, are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role for the synthesis of the menaquinone precursor 1,4-dihydroxy-2-naphthoate (DHNA) in cytosolic survival. Together, these data begin to elucidate adaptations that allow cytosolic pathogens to survive in their intracellular niches.

scholarly journals Atg5-Deficient Mice Infected with Francisella tularensis LVS Demonstrate Increased Survival and Less Severe Pathology in Internal Organs

2020 ◽  
Vol 8 (10) ◽  
pp. 1531
Author(s):  
Ina Kelava ◽  
Mirna Mihelčić ◽  
Mateja Ožanič ◽  
Valentina Marečić ◽  
Maša Knežević ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Internal Organs ◽  
Tularensis Subsp ◽  
Cell Cytosol ◽  
Severe Pathology ◽  
Deficient Mice

Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.

scholarly journals Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

2014 ◽  
Vol 83 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Gael Gesbert ◽  
Elodie Ramond ◽  
Fabiola Tros ◽  
Julien Dairou ◽  
Eric Frapy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Cell Types ◽  
Host Cells ◽  
Metabolic Adaptation ◽  
Amino Acid Transporters ◽  
Loss Of Function ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication ofFrancisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation ofFrancisella. Inactivation of IleP severely impaired intracellularF. tularensissubsp.novicidamultiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of theilePgene inF. tularensispathogenesis, we constructed a chromosomal deletion mutant ofileP(ΔFTL_1803) in theF. tularensissubsp.holarcticalive vaccine strain (LVS). Inactivation of IleP in theF. tularensisLVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication ofFrancisellaand suggest that virulentF. tularensissubspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

scholarly journals CD38 Controls the Innate Immune Response against Listeria monocytogenes

2013 ◽  
Vol 81 (11) ◽  
pp. 4091-4099 ◽  
Author(s):  
Timo Lischke ◽  
Kira Heesch ◽  
Valéa Schumacher ◽  
Michael Schneider ◽  
Friedrich Haag ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Listeria Monocytogenes ◽  
Innate Immune Response ◽  
Cell Activation ◽  
Cell Types ◽  
Innate Immune ◽  
Infection Model ◽  
Content Type ◽  
Inflammatory Monocytes

ABSTRACTCD38, adenosine-5′-diphosphate-ribosyl cyclase 1, is a multifunctional enzyme, expressed on a wide variety of cell types. CD38 has been assigned diverse functions, including generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Using a murineListeria monocytogenesinfection model, we found that CD38 knockout (KO) mice were highly susceptible to infection. Enhanced susceptibility was already evident within 3 days of infection, suggesting a function of CD38 in the innate immune response. CD38 was expressed on neutrophils and inflammatory monocytes, and especially inflammatory monocytes further upregulated CD38 during infection. Absence of CD38 caused alterations of the migration pattern of both cell types to sites of infection. We observed impaired accumulation of cells in the spleen but surprisingly similar or even higher accumulation of cells in the liver. CD38 KO and wild-type mice showed similar changes in the composition of neutrophils and inflammatory monocytes in blood and bone marrow, indicating that mobilization of these cells from the bone marrow was CD38 independent.In vitro, macrophages of CD38 KO mice were less efficient in uptake of listeria but still able to kill the bacteria. Dendritic cells also displayed enhanced CD38 expression following infection. However, absence of CD38 did not impair the capacity of mice to prime CD8+T cells againstL. monocytogenes, and CD38 KO mice could efficiently control secondary listeria infection. In conclusion, our results demonstrate an essential role for CD38 in the innate immune response againstL. monocytogenes.

scholarly journals A Heterologous Prime-Boost Vaccination Strategy Comprising the Francisella tularensis Live Vaccine StraincapBMutant and Recombinant Attenuated Listeria monocytogenes Expressing F. tularensis IglC Induces Potent Protective Immunity in Mice against Virulent F. tularensis Aerosol Challenge

2013 ◽  
Vol 81 (5) ◽  
pp. 1550-1561 ◽  
Author(s):  
Qingmei Jia ◽  
Richard Bowen ◽  
Jacob Sahakian ◽  
Barbara Jane Dillon ◽  
Marcus A. Horwitz
Keyword(s):  
T Cells ◽  
Listeria Monocytogenes ◽  
Survival Rate ◽  
Survival Time ◽  
Francisella Tularensis ◽  
Vaccination Strategy ◽  
Content Type ◽  
Mean Survival Time ◽  
Boost Vaccination ◽  
Ifn Γ

ABSTRACTFrancisella tularensis, the causative agent of tularemia, is a category A bioterrorism agent. A vaccine that is safer and more effective than the currently available unlicensedF. tularensislive vaccine strain (LVS) is needed to protect against intentional release of aerosolizedF. tularensis, the most dangerous type of exposure. In this study, we employed a heterologous prime-boost vaccination strategy comprising intradermally administered LVS ΔcapB(highly attenuatedcapB-deficient LVS mutant) as the primer vaccine and rLm/iglC (recombinant attenuatedListeria monocytogenesexpressing theF. tularensisimmunoprotective antigen IglC) as the booster vaccine. Boosting LVS ΔcapB-primed mice with rLm/iglC significantly enhanced T cell immunity; their splenic T cells secreted significantly more gamma interferon (IFN-γ) and had significantly more cytokine (IFN-γ and/or tumor necrosis factor [TNF] and/or interleukin-2 [IL-2])-producing CD4+and CD8+T cells uponin vitroIglC stimulation. Importantly, mice primed with LVS ΔcapBor rLVS ΔcapB/IglC, boosted with rLm/iglC, and subsequently challenged with 10 50% lethal doses (LD50) of aerosolized highly virulentF. tularensisSchu S4 had a significantly higher survival rate and mean survival time than mice immunized with only LVS ΔcapB(P< 0.0001); moreover, compared with mice immunized once with LVS, primed-boosted mice had a higher survival rate (75% versus 62.5%) and mean survival time during the first 21 days postchallenge (19 and 20 days for mice boosted after being primed with LVS ΔcapBand rLVS ΔcapB/IglC, respectively, versus 17 days for mice immunized with LVS) and maintained their weight significantly better (P< 0.01). Thus, the LVS ΔcapB-rLm/iglC prime-boost vaccination strategy holds substantial promise for a vaccine that is safer and at least as potent as LVS.

scholarly journals Roles of Reactive Oxygen Species-Degrading Enzymes of Francisella tularensis SCHU S4

2015 ◽  
Vol 83 (6) ◽  
pp. 2255-2263 ◽  
Author(s):  
Johan Binesse ◽  
Helena Lindgren ◽  
Lena Lindgren ◽  
Wayne Conlan ◽  
Anders Sjöstedt
Keyword(s):  
Reactive Oxygen Species ◽  
Francisella Tularensis ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Content Type ◽  
Double Deletion ◽  
Genes Encoding ◽  
J774 Cells ◽  
Schu S4 ◽  
Increased Susceptibility

Francisella tularensisis a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion ofFTT0086andkatGshowed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO−) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO−but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4FTT0086gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function.

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