Yousef Abu Kwaik

The modulation of host cell apoptosis by intracellular bacterial pathogens

Recent years have witnessed significant advances in unraveling the elegant mechanisms by which intracellular bacterial pathogens induce and/or block apoptosis, which can influence disease progression. This intriguing aspect of the host-pathogen interaction adds another fascinating dimension to our understanding of the exploitation of host cell biology by intracellular bacterial pathogens.

The use of differential display‐PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages

The differential display (DD)‐PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells. Several DD‐PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post‐infection of the U937 macrophage‐like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage‐induced locus) was induced by intracellular bacteria during the first few hours post‐infection of macrophages but the expression was downregulated by 12 h post‐infection. Transcription of eml was not growth phase‐related in vitro, and was not affected by in vitro stress stimuli. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD‐PCR product was cloned. Six mini‐Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L. pneumophila chromosome. Compared to the wild‐type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100‐fold increase in killing by macrophages during the first 5h of the intracellular infection, and showed a 100‐fold increase in killing during the first 24 h of infection of the amoeba Hartmanella vermiformis. The 6th mutant had a phenotype indistinguishable from the wild‐type strain. The cytopathicity defect of the mutants to the U937 cells was restored to wild‐type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragment containing eml is a novel L. pneumophila locus whose expression is uniquely induced by non‐stress stimuli during early stages of the intracellular infection of phagocytic cells. Expression of this locus is required for survival of L. pneumophila within macrophages and within amoebae during early stages of the infection.

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages.

ABSTRACT We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.

Hijacking of apoptotic pathwaysby bacterial pathogens.

Increasing evidence indicates that apoptosis of the host cell may constitute a defense mechanism to confine the infection by bacterial pathogens. Certain pathogens have developed elegant mechanisms to modulate the fate of the host cell, which include induction or blockage of apoptosis. These studies will promote our understanding of the pathogenesis of infectious diseases and aid the development of means for therapeutic intervention.

Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant

Expression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli. Transcription of gspA is regulated by two promoters, one of which is regulated by the σ32 heat‐shock transcription factor. We utilized a gspA promoter fusion to a promoterless lacZ to probe the phagososmal ‘microenvironment’ for the kinetics of exposure of intracellular L. pneumophila to stress stimuli. Expression through the gspA promoter was constitutively induced by approx. 16‐fold throughout the intracellular infection, and occurred predominantly through the σ32‐regulated promoter. Expression of the gspA promoter was induced approx. 4.5‐fold, 5‐, 11‐ and 9‐fold upon exposure of L. pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively. An isogenic insertion mutant of L. pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild‐type strain AA200. Compared to in vitro‐grown wild‐type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli. The wild‐type phenotypes were restored to strain AA224 by complementation with a plasmid containing wild‐type gspA. There was no difference between the wild‐type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae. However, compared to in vitro‐grown bacteria, macrophage‐grown and amoebae‐grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli. Our data showed that regardless of the capacity of L. pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L. pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection. Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage‐induced and stress‐induced proteins within the intracellular environment.

Fatal attraction of mammalian cells to Legionella pneumophila

Legionella pneumophila is a protozoan parasite that causes Legionnaires disease. Its ability to do so is dependent on its capacity to replicate intracellularly within a phagosome that is not trafficked through the endosomal–lysosomal pathway and is surrounded by the rough endoplasmic reticulum. Within this unique niche, the bacterium undergoes alterations in gene expression. In addition, many virulence‐related phenotypes that are induced in vitro by starvation are expressed intracellularly as the bacteria exit the logarithmic growth phase. (p)ppGpp appears to signal expression of the virulence‐related genes in L. pneumophila upon starvation. This growth phase‐dependent phenotypical transition is concomitant with lysis of the host cell, in which both necrosis and apoptosis seem to play roles. Many genetic loci that are required for intracellular replication within mammalian and protozoan cells have been identified, and the majority of them are novel. Two secretion systems have been identified, one of which may be distantly related to type IV secretion systems. The other is a type II secretion system similar to the PilBCD piliation system of Pseudomonas aeruginosa.

icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells.

ABSTRACT The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.

The Cytochrome c Maturation Locus of Legionella pneumophila Promotes Iron Assimilation and Intracellular Infection and Contains a Strain-Specific Insertion Sequence Element

ABSTRACT Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.

Invasion of Mammalian and Protozoan Cells by Legionella pneumophila

The first recognized outbreak of pneumonia due to Legionella pneumophila occurred in Philadelphia in July of 1976 among 180 persons attending the 56th annual American Legion Convention. Twenty nine patients died, and the disease became known as Legionnaires’ disease (Fraser et al., 1977). Guinea pigs were infected with postmortem lung tissue from the patients with fatal Legionnaires’ disease, and embryonated yolk sacs were inoculated with spleen homogenates from the infected guinea pigs. In January of 1977, a gram-negative bacterium was isolated and designated L. pneumophila (McDade et al., 1977). Antisera were subsequently generated which facilitated identification of many previous outbreaks of febrile respiratory illness of unknown etiology that occurred since 1965. The source of the infection during the Legionnaires’ convention was later found to be the air conditioning system in the hotel. It has been documented that the hallmark of Legionnaires’ disease is the intracellular replication of L. pneumophila in the alveolar spaces. At least another 39 species of legionellae have been identified, some of which are associated with disease while others are environmental isolates and whether they can cause disease is not known. L. pneumophila is responsible for more than 80% of cases of Legionnaires’ disease, and among the 13 serogroups of L. pneumophila, serogroup 1 is responsible for more than 95% of Legionnaires’ disease cases. It is estimated that L. pneumophila is responsible for at least 25,000 cases of pneumonia/year in the US. In 1980, Rowbotham described the ability of L. pneumophila to multiply intracellularly within protozoa (Rowbotham, 1980). Since then, L. pneumophila has been described to multiply in many species of protozoa, and this host-parasite interaction is central to the pathogenesis and ecology of L. pneumophila. Intracellular replication of L. pneumophila within mammalian and protozoan cells has been shown to occur in a ribosome-studded phagosome that does not fuse to lysosomes. Fields had hypothesized that the L. pneumophila phagosome fuses to the rough endoplasmic reticulum (RER) (Fields, 1993). Immunocytochemistry has proven this prediction by demonstrating the presence of an RER-specific chaperon, the Bip protein, in the ribosome-studded phagosome within macrophages (Swanson and Isberg, 1995), and protozoa (Abu Kwaik, 1996). Based on these characteristics the L. pneumophila phagosome may be accurately described as endosomal maturation-blocked (EMB) phagosome.

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.