Marina Šantić

Amoebae as Training Grounds for Intracellular Bacterial Pathogens

Free-living amoebae are important predators that control microbial communities. They are ubiquitous and have been isolated from various natural sources such as soil, freshwater, salt water, dust, and air. Although their abundance in soil is only limited, they have been implicated in the stimulation

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm

The Francisella tularensis subsp. novicida‐containing phagosome (FCP) matures into a late endosome‐like stage that acquires the late endosomal marker LAMP‐2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte‐derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80–90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP‐2 similar to the wild‐type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA‐gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.

Host Proteasomal Degradation Generates Amino Acids Essential for Intracellular Bacterial Growth

The bacterial pathogen Legionella pneumophila ensures amino acid supplies by promoting degradation of target host proteins. Legionella pneumophila proliferates in environmental amoeba and human cells within the Legionella-containing vacuole (LCV). The exported AnkB F-box effector of L. pneumophila is anchored into the LCV membrane by host-mediated farnesylation. Here, we report that host proteasomal degradation of Lys48-linked polyubiquitinated proteins, assembled on the LCV by AnkB, generates amino acids required for intracellular bacterial proliferation. The severe defect of the ankB null mutant in proliferation within amoeba and human cells is rescued by supplementation of a mixture of amino acids or cysteine, serine, pyruvate, or citrate, similar to rescue by genetic complementation. Defect of the ankB mutant in intrapulmonary proliferation in mice is rescued upon injection of a mixture of amino acids or cysteine. Therefore, Legionella promotes eukaryotic proteasomal degradation to generate amino acids needed as carbon and energy sources for bacterial proliferation within evolutionarily distant hosts.

Modulation of biogenesis of the Francisella tularensis subsp novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-gamma

Francisella tularensis is a highly virulent facultative intracellular pathogen that has been categorized as a class A bioterrorism agent, and is classified into four subsp, tularensis, holarctica, mediasiatica and novicida. Although the ability of F. tularensis subsp. novicida to cause tularemia in mice is similar to the virulent subsp. tularensis and holarctica, it is attenuated in humans. It is not known whether attenuation of F. tularensis subsp. novicida in humans is resulting from a different route of trafficking within human macrophages, compared with the tularensis or holarctica subsp. Here we show that in quiescent human monocytes‐derived macrophages (hMDMs), the F. tularensis subsp. novicida containing phagosome (FCP) matures into a late endosome‐like stage that acquires the late endosomal marker LAMP‐2 but does not fuse to lysosomes. This modulation of phagosome biogenesis by F. tularensis is followed by disruption of the phagosome at 4–12 h and subsequent bacterial escape into cytoplasm where the organism replicates. In IFN‐γ‐activated hMDMs, intracellular replication of F. tularensis is completely inhibited, and is associated with failure of the organism to escape from the phagosome into the cytoplasm for up to 24 h after infection. In IFN‐γ‐activated hMDMs, the FCPs acquire the lysosomal enzymes Cathepsin D, which is excluded in quiescent hMDMs. When the lysosomes of IFN‐γ‐activated hMDMs are preload with Texas Red Ovalbumin or BSA‐gold, the FCPs acquire both lysosomal tracers. In contrast, both lysosomal tracers are excluded from the FCPs within quiescent hMDMs. We conclude that although F. tularensis subsp. novicida is attenuated in humans, it modulates biogenesis of its phagosome into a late endosome‐like compartment followed by bacterial escape into the cytoplasm within quiescent hMDMs, similar to the virulent subsp. tularensis. In IFN‐γ‐activated hMDMs, the organism fails to escape into the cytoplasm and its phagosome fuses to lysosomes, similar to inert particles.

Acquisition of the Vacuolar ATPase Proton Pump and Phagosome Acidification Are Essential for Escape of Francisella tularensis into the Macrophage Cytosol

ABSTRACT The Francisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), ∼90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring the iglD mutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of the iglD mutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of bacterial escape into the cytosol is completely reversible, as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late-endosomal fusion regulator Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.

A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol

Francisella tularensis is an intracellular bacterial pathogen, and is a category A bioterrorism agent. Within quiescent human macrophages, the F. tularensis pathogenicity island (FPI) is essential for bacterial growth within quiescent macrophages. The F. tularensis‐containing phagosome matures to a late endosome‐like stage that does not fuse to lysosomes for 1–8 h, followed by gradual bacterial escape into the macrophage cytosol. Here we show that the FPI protein IglD is essential for intracellular replication in primary human monocyte‐derived macrophages (hMDMs). While the parental strain replicates robustly in pulmonary, hepatic and splenic tissues of BALB/c mice associated with severe immunopathologies, the isogenic iglD mutant is severely defective. Within hMDMs, the iglD mutant‐containing phagosomes mature to either a late endosome‐like phagosome, similar to the parental strain, or to a phagolysosome, similar to phagosomes harbouring the iglC mutant control. Despite heterogeneity and alterations in phagosome biogenesis, the iglD mutant bacteria escape into the cytosol faster than the parental strain within hMDMs and pulmonary cells of BALB/c mice. Co‐infections of hMDMs with the wild‐type strain and the iglD mutant, or super‐infection of iglD mutant‐infected hMDMs with the wild‐type strain show that the mutant strain replicates robustly within the cytosol of hMDMs coinhabited by the wild strain. However, when the wild‐type strain‐infected hMDMs are super‐infected by the iglD mutant, the mutant fails to replicate in the cytosol of communal macrophages. This is the first demonstration of a F. tularensis novel protein essential for proliferation in the macrophage cytosol. Our data indicate that F. tularensis transduces signals to the macrophage cytosol to remodel it into a proliferative niche, and IglD is essential for transduction of these signals.

Incomplete Activation of Macrophage Apoptosis during Intracellular Replication of Legionella pneumophila

ABSTRACT The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

Cell biology and molecular ecology of Francisella tularensis.

Francisella tularensis is a highly infectious intracellular bacterium that causes the fulminating disease tularemia, which can be transmitted between mammals by arthorpod vectors. Genomic studies have shown that the F. tularensis has been undergoing genomic decay with the most virulent strains having the lowest number of functional genes. Entry of F. tularensis into macrophages is mediated by looping phagocytosis and is associated with signalling through Syk tyrosine kinase. Within macrophages and arthropod‐derived cells, the Francisella‐containing phagosome matures transiently into an acidified late endosome‐like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol within 30–60 min, and bacterial proliferation within the cytosol. The Francisella pathogenicity island, which potentially encodes a putative type VI secretion system, is essential for phagosome biogenesis and bacterial escape into the cytosol within macrophages and arthropod‐derived cells. Initial sensing of F. tularensis in the cytosol triggers IRF‐3‐dependent IFN‐β secretion, type I IFNR‐dependent signalling, activation of the inflammasome mediated by caspase‐1, and a pro‐inflammatory response, which is suppressed by triggering of SHIP. The past few years have witnessed a quantum leap in our understanding of various aspects of this organism and this review will discuss these remarkable advances.

Maturation of the Legionella pneumophila-Containing Phagosome into a Phagolysosome within Gamma Interferon-Activated Macrophages

ABSTRACT Legionella pneumophila is an intracellular pathogen that modulates the biogenesis of its phagosome to evade endocytic vesicle traffic. The Legionella-containing phagosome (LCP) does not acquire any endocytic markers and is remodeled by the endoplasmic reticulum during early stages. Here we show that intracellular replication of L. pneumophila is inhibited in gamma interferon (IFN-γ)-activated, bone marrow-derived mouse macrophages and IFN-γ-activated, human monocyte-derived macrophages in a dose-dependent manner. This inhibition of intracellular replication is associated with the maturation of the LCP into a phagolysosome, as documented by the acquisition of LAMP-2, cathepsin D, and lysosomal tracer Texas Red ovalbumin, and with the failure of the LCP to be remodeled by the rough endoplasmic reticulum. We conclude that IFN-γ-activated macrophages override the ability of L. pneumophila to evade endocytic fusion and that the LCP is processed through the “default” endosomal-lysosomal degradation pathway.

Intracellular fate of Francisella tularensis within arthropod-derived cells.

Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster-derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica-derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.