Metal complexes of lactoferrin and their effect on the intracellular multiplication of Legionella pneumophila

The action of bovine lactoferrin saturated with iron, zinc and manganese on the intracellular multiplication of Legionella pneumophila in HeLa cells has been tested. The results obtained showed that lactoferrin did not influence the invasive efficiency of Legionella. The intracellular multiplication of the bacterium was inhibited by apo-lactoferrin and by lactoferrin saturated with manganese and zinc, whereas lactoferrin saturated with iron enhanced the intracellular growth. Experiments in parallel were performed with iron, manganese and zinc citrate to test the effect due to the metal ions alone. Even in this condition the addition of an iron chelate enhanced the multiplication of Legionella while the manganese chelate produced a certain inhibition.     

The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages

Legionella pneumophila, the causative agent of Legionnaires’ disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular lifestyle is the ability of the organism to replicate within a specialized phagosome which does not fuse with Iysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome–Iysosome fusion. In a previous study, a 12kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-Iysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-Iysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1–2.4kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have Inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.     

Autophagy induced by 2-deoxy-D-glucose suppresses intracellular multiplication of Legionella pneumophila in A/J mouse macrophages

Legionella pneumophila Philadelphia-1 (Lp-1) can grow intracellularly in A/J mouse peritoneal macrophages (A/J Mφ). We previously reported that 2-deoxy-D-glucose (2dG), when added in macrophage culture media, inhibited the intracellular multiplication of Lp-1 in A/J Mφ. We found that 1mM of 2dG caused LC3-II-conversion that reflects an induction of autophagy and that 1 and 10mM of 2dG induced apoptosis associated with caspase-4 activation. We therefore investigated whether 2dG-induced autophagy or apoptosis suppresses the replication of Lp-1 in 2dG-treated A/J Mφ. When autophagy-related 5 (Atg5) was knocked down by RNA interference, the Atg5-siRNA-transfected cells revealed an enhanced replication of Lp-1 in A/J Mφ compared with the nontargetting siRNA-transfected cells. However, caspase-4 inhibitor did not affect the 2dG-induced inhibition of intracellular multiplication of Lp-1 in A/J Mφ. These findings suggested that autophagy, not apoptosis, suppressed the intracellular growth of Lp-1 in A/J Mφ when 1 or 10 mM of 2dG were added to the culture media.     

Sequence Polymorphism of dotA and mip Alleles Mediating Invasion and Intracellular Replication of Legionella pneumophila

Legionella pneumophila inhabit a variety of natural and man-made aquatic environments, where they live primarily as intracellular parasites of protozoans. Given the proper exposure, however, they can cause opportunistic pneumonic infections in humans. The products of two L. pneumophila genes, dotA and mip, are part of the mechanism mediating the initial invasion of eukaryotic cells, and subsequent intracellular survival and multiplication. In this study, DNA polymorphism of the dotA and mip genes was assessed for 17 clinical and environmental isolates by nucleotide sequencing to determine the level of sequence variation, rates of molecular evolution, and history of gene divergence. The mip gene is highly conserved, whereas dotA is extremely variable, with an average level of nucleotide diversity four times greater than that of mip. Gene trees for each locus support a division of the L. pneumophila isolates into two clonal lineages. There are several disagreements between the gene trees suggesting that although L. pneumophila has a clonal population structure, genetic exchange has contributed to genotypic variation among strains in nature. Received: 12 July 2001 / Accepted: 20 August 2001     

Impact of Non-Legionella Bacteria on the Uptake and Intracellular Replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis

In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.     

Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

Legionnaires' disease is an emerging, severe, pneumonia‐like illness caused by the Gram‐negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K⁺ transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K⁺ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella‐containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild‐type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K⁺ transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K⁺ transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient‐limited conditions.     

Inhibitory Effects of 2-Deoxy-D-glucose on Methanol Metabolism in Torulopsis sp.

Inhibitory effects of 2-deoxy-D-glucose (2dG) on methanol metabolism in Torulopsis A–12 were investigated. The remarkable inhibition was observed on growth in the presence of 50 μg/ml of 2dG when methanol was used as a carbon source. At the earlier time of incubation with 2dG, the incorporation of ¹⁴C-methanol into hexose-phosphate by intact cells was inhibited by 2dG, and this led to the reduction of the intracellular concentration of hexose-phosphates. In the later period of incubation the specific activity of alcohol oxidase reduced, and formate accumulated extracellularly.     

Influence of Acanthamoeba castellanii on Intracellular Growth of Different Legionella Species in Human Monocytes

Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449–2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.     

Inhibitors of cell division and protoplasmic streaming fail to cause a detectable effect on intracellular calcium levels in stamen-hair cells of Tradescantia virginiana L.

The herbicides amiprophos-methyl (APM) and oryzalin disrupt mitosis and cytokinesis in plant cells by causing the depolymerization of microtubules. These drugs have also been shown to affect calcium sequestration by mitochondria. Controversy thus exists as to whether microtubule depolymerization occurs as a result of direct interaction between the drug and tubulin, or because of elevated intracellular calcium levels resulting from drug interference with calcium regulation. In order to clarify this issue we have directly measured the effect of these herbicides and other cell-motility-altering drugs on intracellular calcium levels in stamen-hair cells of Tradescantia. The results indicate that low levels (1–3 M) of APM and oryzalin can act within 3–7 min causing disorganization of mitosis. Studies using the calcium indicator indo-1 injected into stamen-hair cells to monitor internal levels of calcium, show that at drug concentrations where inhibitory effects on mitosis and-or cytokinesis are clearly seen, APM, oryzalin, isopropyl-N-phenyl carbamate, caffeine and cytochalasin D produce no change in intracellular calcium levels. Furthermore, except for cytochalasin D, these drugs do not inhibit cytoplasmic streaming, a calcium-sensitive process. We conclude that the mode of action of these drugs on the cytoskeleton is independent of an effect on intracellular calcium.Abbreviations and Symbols APM amiprophos-methyl - [Ca²⁺]_i free intracellular calcium ion concentration - CD cytochalasin D - DMSO dimethylsulfoxide - IPC isopropyl N-phenylcarbamate - MT(s) microtubule(s) To whom correspondence should be addressedWe thank Dr. L.C. Morejohn, University of Texas, Austin, for encouraging us to perform this study and for his gift of amiprophosmethyl and oryzalin. We also thank our colleagues at the University of Massachusetts for many helpful discussions. This work has been supported by grants from the U.S. Department of Agriculture (88-37261-3727) and the National Science Foundation (DCB-88-01-01750).     

High-Resolution Linkage Map of Mouse Chromosome 13 in the Vicinity of the Host Resistance LocusLgn1

Natural resistance of inbred mouse strains to infection withLegionella pneumophilais controlled by the expression of a single dominant gene on chromosome 13, designatedLgn1.The genetic difference atLgn1is phenotypically expressed as the presence or absence of intracellular replication ofL. pneumophilain host macrophages. In our effort to identify theLgn1gene by positional cloning, we have generated a high-resolution linkage map of theLgn1chromosomal region. For this, we have carried out extensive segregation analysis in a total of 1270 (A/J × C57BL/6J) × A/J informative backcross mice segregating the resistance allele of C57BL/6J and the susceptibility allele of A/J. Additional segregation analyses were carried out in three preexisting panels of C57BL/6J ×Mus spretusinterspecific backcross mice. A total of 39 DNA markers were mapped within an interval of approximately 30 cM overlapping theLgn1region. Combined pedigree analyses for the 5.4-cM segment overlappingLgn1indicated the locus order and the interlocus distances (in cM):D13Mit128–(1.4)–D13Mit194–(0.1)–D13Mit147–(0.9)–D13Mit36–(0.9)–D13Mit146–(0.2)–Lgn1/D13Mit37–(1.0)–D13Mit70.Additional genetic linkage studies of markers not informative in the A/J × C57BL/6J cross positionedD13Mit30, -72, -195,and-203, D13Gor4, D13Hun35,andMtap5in the immediate vicinity of theLgn1locus. The marker density and resolution of this genetic linkage map should allow the construction of a physical map of the region and the isolation of YAC clones overlapping the gene.     

Legionella pneumophila utilizes a single‐player disulfide‐bond oxidoreductase system to manage disulfide bond formation and isomerization

Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein‐disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S‐S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared with E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant‐negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single‐player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.     

Natural resistance to infection with Legionella pneumophila: chromosomal localization of the Lgn1 susceptibility gene

Legionella pneumophila is a strict intracellular pathogen that replicates in the professional phagocytes of the human and guinea pig host. Although murine macrophages from most inbred strains are non-permissive to intracellular replication of L. pneumophila, inflammatory macrophages from the mouse strain A/J are completely permissive to intracellular replication of this bacterium. This genetic difference is controlled by the expression of a single autosomal gene designated Lgn1, with non-permissiveness behaving as completely dominant over permissiveness. We have used a total of 25 AXB/BXA recombinant inbred mouse strains and 182 (A/JxC57BL/6J)xA/J segregating backcross progeny (A/J, permissive; C57BL/6J, non-permissive) to map the Lgn1 gene. Animals were individually type for tolerance to intracellular replication by in vitro infection of their inflammatory macrophages with L. pneumophila. All animals segregated into two non-overlapping groups. Examination of the strain distribution pattern of the AXB/BXA strains for Lgn1 initially identified linkage to Chromosome (Chr) 13 markers. Genotyping of the 25 AXB/BXA strains and the 182 backcross progeny for 11 Chr 13 markers established that Lgn1 mapped to Chr 13, with the gene order and intergene distance D13Mit231-(5.5±1.5)-D13Mit193-(2.2±0.9)-D13Mit194-(1.1±0.6)-D13Mit128-(2.6±1.0)-Lgn1-(2.2±0.9)-D13Mit70-(3.9±1.3)-D13Mit73-(7.2±1.7)-D13Mit53-(0.7±0.5)-D13Mit32-(0.7±0.5)-D13Mit77-(0.7±0.5)-D13Mit78. This portion of Chr 13 is homologous to the distal portion of human Chr 5, 5q11–5q13, suggesting a possible location of a human LGN1 homolog. Understanding the molecular basis of the high permissiveness of A/J macrophage to L. pneumophila may shed light on the survival strategy of this bacterium in highly permissive human phagocytes. This may be achieved by positional cloning of Lgn1, and the identification of the Lgn1 subchromosomal region reported here is a first step towards that goal.     

The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.     

Effect of cytochalasins on surfactant release from alveolar type II cells

Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.     

Replication of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> in Floating Biofilms

Biofilms are a major source of human pathogenic Legionella pneumophila in aquatic systems. In this study, we investigated the capacity of L. pneumophila to colonize floating biofilms and the impact of Acanthamoeba castellanii on the replication of biofilm-associated Legionella. Biofilms were grown in Petri dishes and consisted of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve, and Pseudomonas aeruginosa. Six hours following inoculation, Legionella were detected in floating biofilms in mean concentrations of 1.4 × 10⁴ cells/cm² (real-time polymerase chain reaction) and 8.3 × 10² CFU/cm² (culture). Two-way analysis of variance tests and fluorescent in situ hybridization clearly proved that increased biofilm-associated L. pneumophila concentrations were the result of intracellular replication in A. castellanii. Forty-eight hours after the introduction of A. castellanii in the Petri dishes, 90 ± 0.8% of the amoebae (infection rate) were completely filled with highly metabolic active L. pneumophila (mean infection intensity).     

Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones

Legionella species are ubiquitous, waterborne bacteria that thrive in numerous ecological niches. Yet, in contrast to many other environmental bacteria, Legionella spp. are also able to grow intracellularly in predatory protozoa. This feature mainly accounts for the pathogenicity of Legionella pneumophila, which causes the majority of clinical cases of a severe pneumonia termed Legionnaires' disease. The pathomechanism underlying L. pneumophila infection is based on macrophage resistance, which in turn is largely defined by the opportunistic pathogen's resistance towards amoebae. L. pneumophila replicates in macrophages or amoebae in a unique membrane‐bound compartment, the Legionella‐containing vacuole (LCV). LCV formation requires the bacterial intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and involves a plethora of translocated effector proteins, which subvert pivotal processes in the host cell. Of the ca. 300 different experimentally validated Icm/Dot substrates, about 50 have been studied and attributed a cellular function to date. The versatility and ingenuity of these effectors' mode of actions is striking. In this review, we summarize insight into the cellular functions and biochemical activities of well‐characterized L. pneumophila effector proteins and the host pathways they target. Recent studies not only substantially increased our knowledge about pathogen–host interactions, but also shed light on novel biological mechanisms.     

Cytokines and legionellosis

Recent studies have led to an enhanced understanding of the role of cell-mediated immunity and cytokines in Legionnaires' disease. In particular, the effect of interferon gamma on human mononuclear phagocyte iron metabolism and the role of iron availability inLegionella pneumophila intracellular multiplication in human monocytes has been elucidated. With this knowledge it is now possible to develop treatment strategies for Legionnaires' disease using interferon gamma and/or agents affecting human mononuclear phagocyte iron metabolism.Abbreviations M-CSF Macrophage colony stimulating factor