Miljenko Dorić

Temporal Pore Formation-Mediated Egress from Macrophages and Alveolar Epithelial Cells by Legionella pneumophila

ABSTRACT Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79–90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are “trapped” within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.

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.

HtrA Homologue of Legionella pneumophila: an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

ABSTRACT Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103➤R and S212➤A) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrAmutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrApromoter to probe the phagosomal microenvironment harboringL. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotAmutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.

Murine model of pregnancy‐associated Listeria monocytogenes infection

Listeria monocytogenes has been recognized as a significant pathogen, occurring worldwide, capable of causing animal and human infections. In its most severe form, listeriosis is an invasive disease that affects immunocompromised patients. Additionally, pregnant women represent a high-risk group for L. monocytogenes infection. Abortion, stillbirth or severe neonatal infection can be the serious outcome of such an infection. In an experimental murine model of pregnancy-associated listeriosis we studied the impact of L. monocytogenes on the maternal immune response and pregnancy outcome. In comparison to virgin animals, pregnant mice mounted lower levels of protective cytokines and were unable to eliminate the pathogen. The impaired maternal immune response that has been found both on the systemic and local level, facilitated bacterial multiplication in the liver, placenta and ultimately in the fetal tissues. This resulted in severe necrotizing hemorrhagic hepatitis and Listeria-induced placental necrosis, increasing the incidence of postimplantation loss and poor pregnancy outcome.

Plasma cytokine response in mice with bacterial infection

BACKGROUND: Exposure to microorganisms elicts the production of cytokines. These soluble factors enhance several innate immune functions and regulate the ensuing specific immune response aimed at limiting the spread of infection. AIM: This study was undertaken to quantify the plasma levels of pro-inflammatory cytokines during the course of primary Listeria monocytogenes and Campylobacter jejuni infection. Using an in vivo infection the relationship between endogenous cytokines and the bacterial number in the liver of infected animals was examined. METHODS: C57BL/6 mice were infected by the intraperitoneal route. At different time points we determined the number of colony-forming units of bacteria in the liver of infected animals and paralled these with the plasma levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) measured by enzyme immunoassays. RESULTS: L. monocytogenes infection lasted 10-11 days. IFN-gamma production occurred in the early phase but was more pronounced after day 4, following the appearance of specific immunity. The duration of experimental campylobacteriosis was 15 days. Early IFN-gamma production was not significant but a progressive rise of this cytokine in plasma was seen during the second week post infection. Mice produced measurable amounts of plasma TNF-alpha immediately after being given viable L. monocytogenes, peaking on day 2-3 when the greatest number of bacteria was present in the examined organs. During C. jejuni infection plasma TNF-alpha was produced in a similar manner, but the highest concentrations were found a few days later than in listeriosis, in correlation with the different course of campylobacteriosis. The quantity of IL-6 increased and decreased in concordance with clearance of L monocytogenes and the clinical status of the animals. C. jejuni did not promote the induction of this cytokine. This is to some extent an unusual finding. With respect to the role of IL-6 in Th2 responses and antibody production, the appearance of this cytokine in campylobacteriosis was more expected. DISCUSSION: During systemic bacterial infection, a network of pro-inflammatory cytokines is activated and blood levels of these cytokines are elevated, albeit inconsistently, with large individual variations and depending on microbial characteristics and structure.

Host-Dependent Trigger of Caspases and Apoptosis by Legionella pneumophila

ABSTRACT The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.

The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga

We have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation‐mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) ΔG544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C‐terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation‐mediated cytolysis. The icmT wild‐type allele complemented the icmT null mutant for both defects of intracellular replication and pore formation‐mediated cytolysis and egress from mammalian cells. In contrast, the icmTΔG544 allele complemented the icmT null mutant for intracellular growth, but not for the pore‐forming activity. Consistent with their defect in pore formation‐mediated cytotoxicity in vitro, both mutants failed to cause pulmonary inflammation in A/J mice. Interestingly, the rib mutant was severely defective in intracellular growth within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy confirmed that the rib mutant and the icmT null mutant were severely and completely defective, respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C‐terminus domain of IcmT is essential for the pore‐forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells.

Experimental Legionella longbeachae infection in intratracheally inoculated mice

This study established an experimental model of replicative Legionella longbeachae infection in A/J mice. The animals were infected by intratracheal inoculation of 10(3)-10(9) c.f.u. L. longbeachae serogroup 1 (USA clinical isolates D4968, D4969 and D4973). The inocula of 10(9), 10(8), 10(7) and 10(6) c.f.u. of all tested L. longbeachae serogroup 1 isolates were lethal for A/J mice. Inoculation of 10(5) c.f.u. L. longbeachae caused death in 90 % of the animals within 5 days, whilst inoculation of 10(4) c.f.u. caused sporadic death of mice. All animals that received 10(3) c.f.u. bacteria developed acute lower respiratory disease, but were able to clear Legionella from the lungs within 3 weeks. The kinetics of bacterial growth in the lungs was independent of inoculum size and reached a growth peak about 3 logarithms above the initial inoculum at 72 h after inoculation. The most prominent histological changes in the lungs were observed at 48-72 h after inoculation in the form of a focal, neutrophil-dominant, peribronchiolar infiltration. The inflammatory process did not progress towards the interstitial or alveolar spaces. Immunohistological analyses revealed L. longbeachae serogroup 1 during the early phase of infection near the bronchiolar epithelia and later co-localized with inflammatory cells. BALB/c and C57BL/6 mice strains were also susceptible to infection with all L. longbeachae serogroup 1 strains tested and very similar changes were observed in the lungs of infected animals. These results underline the infection potential of L. longbeachae serogroup 1, which is associated with high morbidity and lethality in mice.

Primary Listeria monocytogenes infection in gestating mice.

The facultative intracellular Gram-positive bacteriumListeria monocytogenes is a food-borne pathogen of frequently underestimated importance. Pregnant women represent the high-risk group forL. monocytogenes infection. Abortion, stillbirth or neonatal infection can be the serious outcome of such an infection. Recovery from listeriosis, resistance mechanisms of the host and the effect ofL. monocytogenes on fetal development still remain to be fully understood. The results of our experiments showed an increased susceptibility of gestating BALB/c mice to primaryL. monocytogenes infection. The duration of listeriosis in gestating animals was almost twice longer than in the control group. Furthermore, it was clearly shown that the detrimental effect ofL. monocytogenes on fetal development was more pronounced if the infection was acquired earlier during gestation.

Lymphocyte subpopulations in the blood and cerebrospinal fluid of multiple sclerosis patients in active disease

ABSTRACT— Lymphocyte subpopulations (total T cells, active T cells and B cells) were simultaneously analyzed in peripheral blood and CSF of MS patients. All patients were in active disease, 3 to 4 weeks after first signs of disease activation appeared. Per cent levels and absolute numbers of examined lymphocyte subpopulations in the blood of MS patients were significantly lower than in healthy controls. In MS, the level of active T lymphocytes was lower in CSF than in peripheral blood. The results support our earlier observations relating to the role of active T lymphocytes in the clinical course of disease.