- Email: [email protected]
Cell biology of Legionella pneumophila
MC2111.QXD
12/14/1999 10:44 AM
Page 30
30
Cell biology of Legionella pneumophila Joseph P Vogel*† and Ralph R Isberg† ‡ Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires’ disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome–lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway. Addresses *Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA; e-mail: [email protected] †Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA ‡e-mail: [email protected] Current Opinion in Microbiology 1999, 2:30–34 http://biomednet.com/elecref/1369527400200030 © Elsevier Science Ltd ISSN 1369-5274 Abbreviations CYE charcoal yeast extract dot defective for organelle trafficking ER endoplasmic reticulum icm intracellular multiplication SMH supplemented Mueller-Hinton
Introduction In the environment, the ubiquitous Gram-negative bacterium Legionella pneumophila is normally found in fresh water sources as an intracellular pathogen of amoebae and in biofilms [1,2]. Humans become infected with L. pneumophila by inhaling aerosols from these contaminated water sources. The bacteria then replicate inside alveolar macrophages causing a severe form of pneumonia called Legionnaires’ disease [3]. The key to L. pneumophila’s virulence is its ability to prevent phagosome–lysosome fusion; mutants defective in this trait are incapable of replicating inside host cells and thus are unable to cause disease [4,5]. Recent work on L. pneumophila has focused on understanding how this organism is able to survive and replicate inside host cells.
Intracellular life cycle of L. pneumophila L. pneumophila is internalized by phagocytic cells via conventional zippering or by a novel uptake process called coiling phagocytosis [6]. In macrophages, uptake appears to be preferentially mediated by complement receptor or by Fc receptor [7,8]. Upon uptake, L. pneumophila is able to alter the macrophage’s endocytic pathway, inhibit acidification of the nascent phagosome and prevent fusion with lysosomes [4,9]. If the organism is successful in altering the endocytic pathway, the phagosome containing the bacteria undergoes a series of maturation events where it sequentially associates with small vesicles, mitochondria and eventually is
surrounded by the rough endoplasmic reticulum (ER) [10,11]. This compartment, called a ‘replicative phagosome’, is the site where the bacteria grow intracellularly [10]. The significance of these cell biological processes is unknown, although a resemblance between the replicative phagosome and an autophagosome has been noted, and stimulation of autophagy partially increases replicative phagosome generation [11]. Nevertheless, it remains unclear if L. pneumophila normally exploits the autophagic pathway, because autophagosomes are surrounded by smooth ER, whereas replicative phagosomes are surrounded by rough ER. Replication inside amoebae appears to be similar to that seen within macrophages. L. pneumophila is able to inhibit phagosome–lysosome fusion within amoebae and the bacteria replicate inside a replicative phagosome surrounded by rough ER [12,13]. One difference between growth in amoebae and macrophages is the host receptor used for attachment and uptake. Because protozoa do not have complement or Fc receptors, they use other receptors for internalizing bacteria. Attachment of L. pneumophila to the protozoa Hartmannella vermiformis has been suggested to be mediated by a Gal/GalNAc lectin receptor, although in Acanthamoeba polyphaga L. pneumophila does not appear to use the same receptor for uptake [14,15]. The receptor used for uptake does not appear to be critical for intracellular growth, since different receptors can be used for uptake into macrophages with no alterations in the capability of growing inside host cells [8]. L. pneumophila is also able to be internalized by nonphagocytic cells including HeLa cells and explanted alveolar epithelial cells [16–18]. Virulent strains of L. pneumophila are reported to be adherent and invasive, whereas nonvirulent strains isolated on SMH plates (described below) remain adherent but are defective for invasion [16,18]. In addition, virulent L. pneumophila are able to replicate inside HeLa cells in a ribosome-studded compartment similar to that seen in phagocytic cells [17,18]. Although nonphagocytic cells can serve as an interesting alternative host for L. pneumophila in the laboratory, results using these cell lines must be interpreted with caution due to the lack of evidence that L. pneumophila normally grows inside nophagocytic cells during the disease process.
Growth phases of L. pneumophila Several reports have described how L. pneumophila’s virulence is effected by different growth conditions [19,20,21•]. Two phases of growth have been described for L. pneumophila. The first, called the ‘multiplicative phase’, is represented by actively multiplying bacteria, such as those found in the early stages of host cell infection and agar-grown bacteria [19]. These bacteria are described as being nonmotile, long and filamentous. The second phase
MC2111.QXD
12/14/1999 10:44 AM
Page 31
Cell biology of Legionella pneumophila Vogel and Isberg
31
Figure 1 The dot/icm genes are located in two 20 kb regions on the L. pneumophila chromosome and may constitute a single pathogenicity island. The genes are named dot for ‘defective in organelle trafficking’ or icm for ‘intracellular multiplication’. The majority of the genes have two designations reflecting their independent identification (dots are shown above the arrows and icms are listed below). Genes coding for proteins predicted to be localized to the bacterial envelope are shown in grey and black. Genes encoding proteins that are not membrane associated are shown by white arrows. Genes with similarity to genes involved in conjugation systems and/or type IV secretion systems are shown in black and include the following: dotG is related to virB10 of A. tumefaciens and ptlG of B. pertussis, dotB is related to virB11 of A. tumefaciens and ptlH of B. pertussis, and dotM and dotL are related to trbA and trbC from the plasmid R64. CitA/tphA is not required for intracellular growth.
Region I
dotD C B
dotA
1kb icmVWX
Region II
icmF
citA
dotO N P E F
G
tphA
icmB J DCG
E
of growth, or ‘active infective phase’, is made up of highly motile short rods and is associated with later stages of infection and lysis of the host cell. Bacteria in the second phase, such as organisms that have recently lysed out of host cells, are reported to be more invasive for reinfection of host cells than organisms grown on agar [20]. Consistent with these results, L. pneumophila contained in an infected amoebae are more infectious in a mouse infection than bacteria grown on agar [22•]. A related shift in virulence can also be seen with broth-grown organisms where exponential phase L. pneumophila are less infectious than postexponential phase bacteria [21•]. These results suggest that L. pneumophila may undergo a developmental switch, perhaps related to that seen with Chlamydia, where cells are primed for the next round of infection prior to lysing out of the infected host cell.
Factors required for intracellular replication of L. pneumophila A variety of approaches have been taken to identify bacterial factors that permit L. pneumophila to survive and replicate inside host cells. Because L. pneumophila can be cultured outside of host cells on bacteriological media, a major approach to identifying critical factors required for intracellular growth has been to isolate mutants that are unable to replicate inside host cells but are still able to grow on agar plates. The first avirulent strains were isolated by repeatedly passaging a wild-type strain on a suboptimal media, supplemented Mueller-Hinton (SMH) agar [23]. It was later shown that a low concentration of sodium ions in the SMH agar was responsible for inhibiting the growth of wild-type strains (see below) [24]. Horwitz [5] exploited the differential growth characteristics of virulent and avirulent L. pneumophila on SMH agar to isolate an avirulent L. pneumophila strain called
HIJK
L
K LM
O
M
P QRST Current Opinion in Microbiology
25D. Characterization of this mutant revealed that it was unable to grow inside macrophages due to an inability in preventing phagosome–lysosome fusion [5]. This mutant was subsequently complemented for intracellular growth, thereby identifying an operon containing four open reading frames, icmWXYZ (icm for intracellular multiplication) [25,26]. It was noted recently that the icmWXYZ locus actually consists of only three adjacent genes, icmV and icmWX, which are divergently transcribed from each other (Genbank Accession U07354). An independent method was used to isolate mutants by enriching for cells that could survive but could not grow inside macrophages (a thymine-less death enrichment) [27]. Complementation of these mutants revealed one additional gene, dotA (dot for defective for organelle trafficking), that was also required for proper targeting in macrophages [28]. The dotA gene is located downstream from icmV and the two genes appear to be in an operon. These four genes have no similarity to genes in the published databases, although three of the genes (dotA, icmV and icmX) are predicted to encode proteins localized to the bacterial envelope. Consistent with this, the DotA protein was shown to be localized to the inner membrane of L. pneumophila [29]. A number of laboratories have made serious efforts over the past few years to isolate L. pneumophila mutants that are defective for intracellular growth. Five additional selections or screens have been published [30–34]. These include a screen of Tn903 insertions for mutants that were unable to kill HL-60-derived human macrophages [30–32], a screen of EMS mutagenized L. pneumophila for mutants unable to form plaques on an A/J macrophage monolayer [33], and a screen of mini-Tn10 insertions for defects in replication within both U937 macrophage-like
MC2111.QXD
12/14/1999 10:44 AM
32
Page 32
Host–microbe interactions: bacteria
cells and A. polyphaga [34]. In addition, two separate enrichments for intracellular mutants were done. The first repeated the thymine-less enrichment with an additional copy of dotA since the original selection only isolated alleles of dotA [35], whereas the second exploited the natural salt sensitivity of wild-type L. pneumophila to enrich for spontaneous salt-resistant mutants unable to replicate in macrophages from an A/J mouse [36]. Although not all of the mutants identified above have been complemented, approximately two dozen dot/icm genes have been identified to date by complementation (Figure 1). None of the mutant selections/screens, however, were saturated so it is possible that additional dot/icm genes exist. The dot/icm genes appear to be transcribed in nine operons and are located in two 20 kb regions on the chromosome [37••,38••]. Sequence analysis of these regions revealed that the dot/icm genes primarily code for proteins predicted to be localized in the envelope and the majority are not homologous to any known genes. Nevertheless, several of Dot/Icm proteins do have sequence similarity to proteins found in bacterial plasmid transfer systems (Figure 1). On the basis of this homology and the ability of L. pneumophila to transfer the mobilizable plasmid RSF1010 from one cell to another in a dot/icm dependent fashion [37••,38••], it was proposed that the dot/icm genes form a secretion system related to the recently described type IV systems [39]. Type IV secretion systems are plasmid transfer systems or are thought to have evolved from such systems and are capable of secreting a wide range of substrates including plasmids, oncogenic DNA, and protein toxins [39].
which have been described as being associated with a functional dot/icm apparatus. These include an ability to transfer the RSF1010 plasmid, a sensitivity to salt, the presence of a contact-dependent cytotoxicity and motility. Salt sensitivity
The ability of L. pneumophila to replicate inside host cells is strongly correlated with a sensitivity to low amounts of sodium chloride. The majority of L. pneumophila mutants unable to replicate inside host cells are also salt resistant. These include a large number of avirulent strains isolated to be salt resistant on SMH agar or on charcoal yeast extract (CYE) plates containing 0.65% sodium chloride [5,36]. In addition, most of the 55 Tn903 mutants identified by Sadosky et al. [30] to be defective for macrophage killing were also resistant to sodium chloride, and all of the mutants identified by screening for loss of plaque forming ability were also salt resistant [33]. Even the original dotA mutant Lp03, isolated via the thymine-less death enrichment, is also salt resistant (JP Vogel, RR Isberg, unpublished data). Furthermore, L. pneumophila in early exponential phase is reported to be both resistant to salt and defective in intracellular survival, whereas late exponential or stationary phase organisms become salt sensitive and more infectious [21•]. One possibility to explain the strong correlation between intracellular replication ability and salt sensitivity is that the dot/icm secretion apparatus is leaky to increased levels of sodium chloride. Inactivation of this machinery would prevent ion flux when the cells are exposed to conditions of elevated sodium levels, and, therefore, salt-resistant strains often are avirulent. Contact-dependent cytotoxicity
The natural substrate(s) secreted by the L. pneumophila dot/icm apparatus has not yet been identified. Based on the homology to type IV secretion systems, it is possible that L. pneumophila is exporting either a nucleic acid, as seen with Agrobacterium tumefaciens, or a protein toxin, as seen with Bordetella pertussis. The dot/icm machinery functions either before or shortly after bacterial uptake to alter the endocytic pathway in macrophages [40•,41•]. In fact, the DotA protein is required for early phagosome trafficking decisions within minutes of bacterial uptake [40•]. As a result, there does not appear to be sufficient time for bacterial DNA to be imported into the macrophage, processed and the encoded products to inhibit normal cell function, as seen with A. tumefaciens import of oncogenic DNA into plant cells. It seems more likely that the L. pneumophila dot/icm machinery is exporting a protein toxin, which can act immediately inside the host cell, similar to that seen with B. pertussis and pertussis toxin.
Additional phenotypes linked to the dot/icm genes Inactivation of the dot/icm genes results in a failure of L. pneumophila to inhibit phagosome–lysosome fusion, thereby preventing intracellular growth. In addition to intracellular growth, there are a number of phenotypes
Wild-type L. pneumophila exhibits a contact-dependent cytotoxicity that results in the rapid lysis of mammalian cells [42]. This cytotoxicity is observed in vitro only when very high levels of bacteria are added to mammalian cells, and it has been shown to be consistent with the creation of pores of defined size in the mammalian membrane [43•]. The cytotoxicity is somehow linked to virulence, because the majority of the dot/icm mutants are no longer cytotoxic [43•]. It does not, however, appear to be biologically relevant for growth in macrophages considering the host cell is lysed before the bacteria has had the opportunity to replicate inside it. Nevertheless, an interesting possibility is that the cytotoxicity is an enhanced reflection of a normal event, the insertion of a pore into the host cell membrane used to mediate transfer of a bacterial substrate(s) into the host cell. Flagella
Previous studies have suggested that there is a physiological link between the presence of flagella and intracellular growth in L. pneumophila. Postexponential phase organisms, which express the virulent phenotype, are motile whereas exponential phase organisms with decreased virulence are not motile [21•], and avirulent mutants isolated on SMH agar are reported to often be nonmotile [44]. Furthermore, a large percentage (70%) of Tn10 insertions that lacked
MC2111.QXD
12/14/1999 10:44 AM
Page 33
Cell biology of Legionella pneumophila Vogel and Isberg
detectable flagellin by colony-blot are also unable to replicate in U937 cells [45]. Motility, however, is not required for growth in macrophages since aflagellar Tn10 insertions were recovered that had no defect in intracellular growth [45]. In addition, a mutant strain lacking flagella due to a defined mutation in a flagellar biosynthetic gene, fliI, was also able to replicate at wild-type levels in U937 cells [46]. As a result, expression of the flagellar structure itself is clearly not required for intracellular growth. The physiological link between motility and the ability to infect macrophages is unknown but it may be due to co-expression of these phenotypes during late exponential/stationary phase.
Other proteins implicated as potentially being involved in intracellular growth of L. pneumophila A variety of other factors have been identified that may play some role in the infectious process. These include a peptidyl-proline isomerase named mip [47], a major protease msp [48,49], and a putative cytotoxin called legiolysin [50]. None of these factors, however, are absolutely required for intracellular growth. In addition, it was recently discovered that L. pneumophila contains a type II secretion system that encodes a type IV pilus [51,52]. L. pneumophila has homologs of the type IV pilus biosynthetic genes pilBCD and a homologue of pilE, the gene coding for a type IV pilin [51,52]. Deletion of these genes results in loss of one type of pilus on the surface of L. pneumophila and a corresponding two fold decrease in adhesion to amoebae and macrophages. Strains lacking the type IV pilus, however, are still able to replicate at wild-type levels in all host cells indicating they are not required for intracellular growth [52]. These and other factors are likely to contribute to the action of the dot/icm genes in allowing L. pneumophila to replicate inside host cells.
Conclusions The past several years have seen major advances in the understanding of how L. pneumophila is able to survive and replicate inside phagocytic cells. The most significant step was the discovery that L. pneumophila uses a type IV secretion system, encoded by the dot/icm genes, to alter the endocytic pathway. The next step will entail the identification of secreted effector molecules and the molecular analysis of their action. Additional fascinating questions include determining if other intracellular pathogens utilize similar systems and investigating the mechanism by which L. pneumophila forms and maintains the replicative phagosome.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Fields BS: The molecular ecology of Legionellae. Trends Microbiol 1996, 4:286-290.
33
2.
Abu Kwaik Y, Gao LY, Stone BJ, Venkataraman C, Harb OS: Invasion of protozoa by Legionella pneumophila and its role in bacterial ecology and pathogenesis. Appl Environ Microbiol 1998, 64:3127-3133.
3.
Horwitz MA, Silverstein SC: Legionnaires’ disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J Clin Invest 1980, 66:441-450.
4.
Horwitz MA: The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med 1983, 158:2108-2126.
5.
Horwitz MA: Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. J Exp Med 1987, 166:1310-1328.
6.
Horwitz MA: Phagocytosis of the Legionnaires’ disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil. Cell 1984, 36:27-33.
7.
Payne NR, Horwitz MA: Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med 1987, 166:1377-1389.
8.
Horwitz MA, Silverstein SC: Interaction of the legionnaires’ disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication. J Exp Med 1981, 153:398-406.
9.
Horwitz MA, Maxfield FR: Legionella pneumophila inhibits acidification of its phagosome in human monocytes. J Cell Biol 1984, 99:1936-1943.
10. Horwitz MA: Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med 1983, 158:1319-1331. 11. Swanson MS, Isberg RR: Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun 1995, 63:3609-3620. 12. Bozue JA, Johnson W: Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion. Infect Immun 1996, 64:668-673. 13. Abu Kwaik Y: The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 1996, 62:2022-2028. 14. Venkataraman C, Haack BJ, Bondada S, Abu Kwaik Y: Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires’ disease bacterium. J Exp Med 1997, 186:537-547. 15. Harb OS, Venkataraman C, Haack BJ, Gao LY, Kwaik YA: Heterogeneity in the attachment and uptake mechanisms of the Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. Appl Environ Microbiol 1998, 64:126-132. 16. Dreyfus LA: Virulence associated ingestion of Legionella pneumophila by HeLa cells. Microb Pathog 1987, 3:45-52. 17.
Cianciotto NP, Stamos JK, Kamp DW: Infectivity of Legionella pneumophila mip mutant for alveolar epithelial cells. Curr Microbiol 1995, 30:247-250.
18. Garduno RA, Quinn FD, Hoffman PS: HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila. Can J Microbiol 1998, 44:430-440. 19. Rowbotham TJ: Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci 1986, 22:678-689. 20. Cirillo JD, Falkow S, Tompkins LS: Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect Immun 1994, 62:3254-3261. 21. Byrne B, Swanson MS: Expression of Legionella pneumophila • virulence traits in response to growth conditions. Infect Immun 1998, 66:3029-3034. This paper describes various conditions that allow enhanced growth within cultured macrophages.
MC2111.QXD
12/14/1999 10:44 AM
34
Page 34
Host–microbe interactions: bacteria
22. Brieland JK, Fantone JC, Remick DG, LeGendre M, McClain M, • Engleberg NC: The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire’s disease. Infect Immun 1997, 65:5330-5333. This paper describes potential phenotypic switch involving virulence traits as a function of growth conditions. 23. McDade JE, Shepard CC: Virulent to avirulent conversion of Legionnaires’ disease bacterium (Legionella pneumophila) — its effect on isolation techniques. J Infect Dis 1979, 139:707-711. 24. Catrenich CE, Johnson W: Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium. Infect Immun 1989, 57:1862-1864.
38. Vogel JP, Andrews HL, Wong SK, Isberg RR: Conjugative transfer •• by the virulence system of Legionella pneumophila. Science 1998, 279:873-876. This paper, along with [37••] shows that the dot/icm genes code for a type IV secretion system used to alter the endocytic pathway of macrophages. 39. Winans SC, Burns DL, Christie PJ: Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol 1996, 4:64-68. 40. Roy CR, Berger KH, Isberg RR: Legionella pneumophila DotA • protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol Microbiol 1998, 28:663-674. See annotation for [41•].
25. Marra A, Blander SJ, Horwitz MA, Shuman HA: Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc Natl Acad Sci USA 1992, 89:9607-9611.
41. Wiater LA, Dunn K, Maxfield FR, Shuman HA: Early events in • phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect Immun 1998, 66:4450-60. This paper and [40•] provide evidence that the dot/icm genes function early during infection of macrophages.
26. Brand BC, Sadosky AB, Shuman HA: The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages. Mol Microbiol 1994, 14:797-808.
42. Husmann LK, Johnson W: Cytotoxicity of extracellular Legionella pneumophila. Infect Immun 1994, 62:2111-2114.
27.
Berger KH, Isberg RR: Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 1993, 7:7-19.
28. Berger KH, Merriam JJ, Isberg RR: Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol 1994, 14:809-822. 29. Roy CR, Isberg RR: Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages. Infect Immun 1997, 65:571-578. 30. Sadosky AB, Wiater LA, Shuman HA: Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect Immun 1993, 61:5361-5373. 31. Purcell M, Shuman HA: The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages. Infect Immun 1998, 66:2245-2255. 32. Segal G, Shuman HA: Characterization of a new region required for macrophage killing by Legionella pneumophila. Infect Immun 1997, 65:5057-5066.
43. Kirby JE, Vogel JP, Andrews HL, Isberg RR: Evidence for pore • forming ability by Legionella pneumophila. Mol Microbiol 1998, 27:323-336. The authors describe the contact-dependent cytotoxicity caused by the dot/icm genes. 44. Elliott JA, Johnson W: Virulence conversion of Legionella pneumophila serogroup 1 by passage in guinea pigs and embryonated eggs. Infect Immun 1982, 35:943-946. 45. Pruckler JM, Benson RF, Moyenuddin M, Martin WT, Fields BS: Association of flagellum expression and intracellular growth of Legionella pneumophila. Infect Immun 1995, 63:4928-4932. 46. Merriam JJ, Mathur R, Maxfield-Boumil R, Isberg RR: Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect Immun 1997, 65:2497-2501. 47.
Cianciotto NP, Eisenstein BI, Mody CH, Toews GB, Engleberg NC: A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect Immun 1989, 57:1255-1262.
33. Andrews HL, Vogel JP, Isberg RR: Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect Immun 1998, 66:950-958.
48. Szeto L, Shuman HA: The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect Immun 1990, 58:2585-2592.
34. Gao LY, Harb OS, Abu Kwaik Y: Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant host cells, mammalian macrophages and protozoa. Infect Immun 1997, 65:4738-4746.
49. Quinn FD, Tompkins LS: Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities. Mol Microbiol 1989, 3:797-805.
35. Swanson MS, Isberg RR: Identification of Legionella pneumophila mutants that have aberrant intracellular fates. Infect Immun 1996, 64:2585-2594.
50. Rdest U, Wintermeyer E, Ludwig B, Hacker J: Legiolysin, a new hemolysin from L. pneumophila. Zentralbl Bakteriol 1991, 274:471-474.
36. Vogel JP, Roy C, Isberg RR: Use of salt to isolate Legionella pneumophila mutants unable to replicate in macrophages. Ann NY Acad Sci 1996, 797:271-272.
51. Liles MR, Viswanathan VK, Cianciotto NP: Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect Immun 1998, 66:1776-1782.
37. ••
Segal G, Purcell M, Shuman HA: Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci USA 1998, 95:1669-1674. See annotation for [38••].
52. Stone BJ, Abu Kwaik Y: Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect Immun 1998, 66:1768-1775.
12/14/1999 10:44 AM
Page 30
30
Cell biology of Legionella pneumophila Joseph P Vogel*† and Ralph R Isberg† ‡ Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires’ disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome–lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway. Addresses *Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA; e-mail: [email protected] †Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA ‡e-mail: [email protected] Current Opinion in Microbiology 1999, 2:30–34 http://biomednet.com/elecref/1369527400200030 © Elsevier Science Ltd ISSN 1369-5274 Abbreviations CYE charcoal yeast extract dot defective for organelle trafficking ER endoplasmic reticulum icm intracellular multiplication SMH supplemented Mueller-Hinton
Introduction In the environment, the ubiquitous Gram-negative bacterium Legionella pneumophila is normally found in fresh water sources as an intracellular pathogen of amoebae and in biofilms [1,2]. Humans become infected with L. pneumophila by inhaling aerosols from these contaminated water sources. The bacteria then replicate inside alveolar macrophages causing a severe form of pneumonia called Legionnaires’ disease [3]. The key to L. pneumophila’s virulence is its ability to prevent phagosome–lysosome fusion; mutants defective in this trait are incapable of replicating inside host cells and thus are unable to cause disease [4,5]. Recent work on L. pneumophila has focused on understanding how this organism is able to survive and replicate inside host cells.
Intracellular life cycle of L. pneumophila L. pneumophila is internalized by phagocytic cells via conventional zippering or by a novel uptake process called coiling phagocytosis [6]. In macrophages, uptake appears to be preferentially mediated by complement receptor or by Fc receptor [7,8]. Upon uptake, L. pneumophila is able to alter the macrophage’s endocytic pathway, inhibit acidification of the nascent phagosome and prevent fusion with lysosomes [4,9]. If the organism is successful in altering the endocytic pathway, the phagosome containing the bacteria undergoes a series of maturation events where it sequentially associates with small vesicles, mitochondria and eventually is
surrounded by the rough endoplasmic reticulum (ER) [10,11]. This compartment, called a ‘replicative phagosome’, is the site where the bacteria grow intracellularly [10]. The significance of these cell biological processes is unknown, although a resemblance between the replicative phagosome and an autophagosome has been noted, and stimulation of autophagy partially increases replicative phagosome generation [11]. Nevertheless, it remains unclear if L. pneumophila normally exploits the autophagic pathway, because autophagosomes are surrounded by smooth ER, whereas replicative phagosomes are surrounded by rough ER. Replication inside amoebae appears to be similar to that seen within macrophages. L. pneumophila is able to inhibit phagosome–lysosome fusion within amoebae and the bacteria replicate inside a replicative phagosome surrounded by rough ER [12,13]. One difference between growth in amoebae and macrophages is the host receptor used for attachment and uptake. Because protozoa do not have complement or Fc receptors, they use other receptors for internalizing bacteria. Attachment of L. pneumophila to the protozoa Hartmannella vermiformis has been suggested to be mediated by a Gal/GalNAc lectin receptor, although in Acanthamoeba polyphaga L. pneumophila does not appear to use the same receptor for uptake [14,15]. The receptor used for uptake does not appear to be critical for intracellular growth, since different receptors can be used for uptake into macrophages with no alterations in the capability of growing inside host cells [8]. L. pneumophila is also able to be internalized by nonphagocytic cells including HeLa cells and explanted alveolar epithelial cells [16–18]. Virulent strains of L. pneumophila are reported to be adherent and invasive, whereas nonvirulent strains isolated on SMH plates (described below) remain adherent but are defective for invasion [16,18]. In addition, virulent L. pneumophila are able to replicate inside HeLa cells in a ribosome-studded compartment similar to that seen in phagocytic cells [17,18]. Although nonphagocytic cells can serve as an interesting alternative host for L. pneumophila in the laboratory, results using these cell lines must be interpreted with caution due to the lack of evidence that L. pneumophila normally grows inside nophagocytic cells during the disease process.
Growth phases of L. pneumophila Several reports have described how L. pneumophila’s virulence is effected by different growth conditions [19,20,21•]. Two phases of growth have been described for L. pneumophila. The first, called the ‘multiplicative phase’, is represented by actively multiplying bacteria, such as those found in the early stages of host cell infection and agar-grown bacteria [19]. These bacteria are described as being nonmotile, long and filamentous. The second phase
MC2111.QXD
12/14/1999 10:44 AM
Page 31
Cell biology of Legionella pneumophila Vogel and Isberg
31
Figure 1 The dot/icm genes are located in two 20 kb regions on the L. pneumophila chromosome and may constitute a single pathogenicity island. The genes are named dot for ‘defective in organelle trafficking’ or icm for ‘intracellular multiplication’. The majority of the genes have two designations reflecting their independent identification (dots are shown above the arrows and icms are listed below). Genes coding for proteins predicted to be localized to the bacterial envelope are shown in grey and black. Genes encoding proteins that are not membrane associated are shown by white arrows. Genes with similarity to genes involved in conjugation systems and/or type IV secretion systems are shown in black and include the following: dotG is related to virB10 of A. tumefaciens and ptlG of B. pertussis, dotB is related to virB11 of A. tumefaciens and ptlH of B. pertussis, and dotM and dotL are related to trbA and trbC from the plasmid R64. CitA/tphA is not required for intracellular growth.
Region I
dotD C B
dotA
1kb icmVWX
Region II
icmF
citA
dotO N P E F
G
tphA
icmB J DCG
E
of growth, or ‘active infective phase’, is made up of highly motile short rods and is associated with later stages of infection and lysis of the host cell. Bacteria in the second phase, such as organisms that have recently lysed out of host cells, are reported to be more invasive for reinfection of host cells than organisms grown on agar [20]. Consistent with these results, L. pneumophila contained in an infected amoebae are more infectious in a mouse infection than bacteria grown on agar [22•]. A related shift in virulence can also be seen with broth-grown organisms where exponential phase L. pneumophila are less infectious than postexponential phase bacteria [21•]. These results suggest that L. pneumophila may undergo a developmental switch, perhaps related to that seen with Chlamydia, where cells are primed for the next round of infection prior to lysing out of the infected host cell.
Factors required for intracellular replication of L. pneumophila A variety of approaches have been taken to identify bacterial factors that permit L. pneumophila to survive and replicate inside host cells. Because L. pneumophila can be cultured outside of host cells on bacteriological media, a major approach to identifying critical factors required for intracellular growth has been to isolate mutants that are unable to replicate inside host cells but are still able to grow on agar plates. The first avirulent strains were isolated by repeatedly passaging a wild-type strain on a suboptimal media, supplemented Mueller-Hinton (SMH) agar [23]. It was later shown that a low concentration of sodium ions in the SMH agar was responsible for inhibiting the growth of wild-type strains (see below) [24]. Horwitz [5] exploited the differential growth characteristics of virulent and avirulent L. pneumophila on SMH agar to isolate an avirulent L. pneumophila strain called
HIJK
L
K LM
O
M
P QRST Current Opinion in Microbiology
25D. Characterization of this mutant revealed that it was unable to grow inside macrophages due to an inability in preventing phagosome–lysosome fusion [5]. This mutant was subsequently complemented for intracellular growth, thereby identifying an operon containing four open reading frames, icmWXYZ (icm for intracellular multiplication) [25,26]. It was noted recently that the icmWXYZ locus actually consists of only three adjacent genes, icmV and icmWX, which are divergently transcribed from each other (Genbank Accession U07354). An independent method was used to isolate mutants by enriching for cells that could survive but could not grow inside macrophages (a thymine-less death enrichment) [27]. Complementation of these mutants revealed one additional gene, dotA (dot for defective for organelle trafficking), that was also required for proper targeting in macrophages [28]. The dotA gene is located downstream from icmV and the two genes appear to be in an operon. These four genes have no similarity to genes in the published databases, although three of the genes (dotA, icmV and icmX) are predicted to encode proteins localized to the bacterial envelope. Consistent with this, the DotA protein was shown to be localized to the inner membrane of L. pneumophila [29]. A number of laboratories have made serious efforts over the past few years to isolate L. pneumophila mutants that are defective for intracellular growth. Five additional selections or screens have been published [30–34]. These include a screen of Tn903 insertions for mutants that were unable to kill HL-60-derived human macrophages [30–32], a screen of EMS mutagenized L. pneumophila for mutants unable to form plaques on an A/J macrophage monolayer [33], and a screen of mini-Tn10 insertions for defects in replication within both U937 macrophage-like
MC2111.QXD
12/14/1999 10:44 AM
32
Page 32
Host–microbe interactions: bacteria
cells and A. polyphaga [34]. In addition, two separate enrichments for intracellular mutants were done. The first repeated the thymine-less enrichment with an additional copy of dotA since the original selection only isolated alleles of dotA [35], whereas the second exploited the natural salt sensitivity of wild-type L. pneumophila to enrich for spontaneous salt-resistant mutants unable to replicate in macrophages from an A/J mouse [36]. Although not all of the mutants identified above have been complemented, approximately two dozen dot/icm genes have been identified to date by complementation (Figure 1). None of the mutant selections/screens, however, were saturated so it is possible that additional dot/icm genes exist. The dot/icm genes appear to be transcribed in nine operons and are located in two 20 kb regions on the chromosome [37••,38••]. Sequence analysis of these regions revealed that the dot/icm genes primarily code for proteins predicted to be localized in the envelope and the majority are not homologous to any known genes. Nevertheless, several of Dot/Icm proteins do have sequence similarity to proteins found in bacterial plasmid transfer systems (Figure 1). On the basis of this homology and the ability of L. pneumophila to transfer the mobilizable plasmid RSF1010 from one cell to another in a dot/icm dependent fashion [37••,38••], it was proposed that the dot/icm genes form a secretion system related to the recently described type IV systems [39]. Type IV secretion systems are plasmid transfer systems or are thought to have evolved from such systems and are capable of secreting a wide range of substrates including plasmids, oncogenic DNA, and protein toxins [39].
which have been described as being associated with a functional dot/icm apparatus. These include an ability to transfer the RSF1010 plasmid, a sensitivity to salt, the presence of a contact-dependent cytotoxicity and motility. Salt sensitivity
The ability of L. pneumophila to replicate inside host cells is strongly correlated with a sensitivity to low amounts of sodium chloride. The majority of L. pneumophila mutants unable to replicate inside host cells are also salt resistant. These include a large number of avirulent strains isolated to be salt resistant on SMH agar or on charcoal yeast extract (CYE) plates containing 0.65% sodium chloride [5,36]. In addition, most of the 55 Tn903 mutants identified by Sadosky et al. [30] to be defective for macrophage killing were also resistant to sodium chloride, and all of the mutants identified by screening for loss of plaque forming ability were also salt resistant [33]. Even the original dotA mutant Lp03, isolated via the thymine-less death enrichment, is also salt resistant (JP Vogel, RR Isberg, unpublished data). Furthermore, L. pneumophila in early exponential phase is reported to be both resistant to salt and defective in intracellular survival, whereas late exponential or stationary phase organisms become salt sensitive and more infectious [21•]. One possibility to explain the strong correlation between intracellular replication ability and salt sensitivity is that the dot/icm secretion apparatus is leaky to increased levels of sodium chloride. Inactivation of this machinery would prevent ion flux when the cells are exposed to conditions of elevated sodium levels, and, therefore, salt-resistant strains often are avirulent. Contact-dependent cytotoxicity
The natural substrate(s) secreted by the L. pneumophila dot/icm apparatus has not yet been identified. Based on the homology to type IV secretion systems, it is possible that L. pneumophila is exporting either a nucleic acid, as seen with Agrobacterium tumefaciens, or a protein toxin, as seen with Bordetella pertussis. The dot/icm machinery functions either before or shortly after bacterial uptake to alter the endocytic pathway in macrophages [40•,41•]. In fact, the DotA protein is required for early phagosome trafficking decisions within minutes of bacterial uptake [40•]. As a result, there does not appear to be sufficient time for bacterial DNA to be imported into the macrophage, processed and the encoded products to inhibit normal cell function, as seen with A. tumefaciens import of oncogenic DNA into plant cells. It seems more likely that the L. pneumophila dot/icm machinery is exporting a protein toxin, which can act immediately inside the host cell, similar to that seen with B. pertussis and pertussis toxin.
Additional phenotypes linked to the dot/icm genes Inactivation of the dot/icm genes results in a failure of L. pneumophila to inhibit phagosome–lysosome fusion, thereby preventing intracellular growth. In addition to intracellular growth, there are a number of phenotypes
Wild-type L. pneumophila exhibits a contact-dependent cytotoxicity that results in the rapid lysis of mammalian cells [42]. This cytotoxicity is observed in vitro only when very high levels of bacteria are added to mammalian cells, and it has been shown to be consistent with the creation of pores of defined size in the mammalian membrane [43•]. The cytotoxicity is somehow linked to virulence, because the majority of the dot/icm mutants are no longer cytotoxic [43•]. It does not, however, appear to be biologically relevant for growth in macrophages considering the host cell is lysed before the bacteria has had the opportunity to replicate inside it. Nevertheless, an interesting possibility is that the cytotoxicity is an enhanced reflection of a normal event, the insertion of a pore into the host cell membrane used to mediate transfer of a bacterial substrate(s) into the host cell. Flagella
Previous studies have suggested that there is a physiological link between the presence of flagella and intracellular growth in L. pneumophila. Postexponential phase organisms, which express the virulent phenotype, are motile whereas exponential phase organisms with decreased virulence are not motile [21•], and avirulent mutants isolated on SMH agar are reported to often be nonmotile [44]. Furthermore, a large percentage (70%) of Tn10 insertions that lacked
MC2111.QXD
12/14/1999 10:44 AM
Page 33
Cell biology of Legionella pneumophila Vogel and Isberg
detectable flagellin by colony-blot are also unable to replicate in U937 cells [45]. Motility, however, is not required for growth in macrophages since aflagellar Tn10 insertions were recovered that had no defect in intracellular growth [45]. In addition, a mutant strain lacking flagella due to a defined mutation in a flagellar biosynthetic gene, fliI, was also able to replicate at wild-type levels in U937 cells [46]. As a result, expression of the flagellar structure itself is clearly not required for intracellular growth. The physiological link between motility and the ability to infect macrophages is unknown but it may be due to co-expression of these phenotypes during late exponential/stationary phase.
Other proteins implicated as potentially being involved in intracellular growth of L. pneumophila A variety of other factors have been identified that may play some role in the infectious process. These include a peptidyl-proline isomerase named mip [47], a major protease msp [48,49], and a putative cytotoxin called legiolysin [50]. None of these factors, however, are absolutely required for intracellular growth. In addition, it was recently discovered that L. pneumophila contains a type II secretion system that encodes a type IV pilus [51,52]. L. pneumophila has homologs of the type IV pilus biosynthetic genes pilBCD and a homologue of pilE, the gene coding for a type IV pilin [51,52]. Deletion of these genes results in loss of one type of pilus on the surface of L. pneumophila and a corresponding two fold decrease in adhesion to amoebae and macrophages. Strains lacking the type IV pilus, however, are still able to replicate at wild-type levels in all host cells indicating they are not required for intracellular growth [52]. These and other factors are likely to contribute to the action of the dot/icm genes in allowing L. pneumophila to replicate inside host cells.
Conclusions The past several years have seen major advances in the understanding of how L. pneumophila is able to survive and replicate inside phagocytic cells. The most significant step was the discovery that L. pneumophila uses a type IV secretion system, encoded by the dot/icm genes, to alter the endocytic pathway. The next step will entail the identification of secreted effector molecules and the molecular analysis of their action. Additional fascinating questions include determining if other intracellular pathogens utilize similar systems and investigating the mechanism by which L. pneumophila forms and maintains the replicative phagosome.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Fields BS: The molecular ecology of Legionellae. Trends Microbiol 1996, 4:286-290.
33
2.
Abu Kwaik Y, Gao LY, Stone BJ, Venkataraman C, Harb OS: Invasion of protozoa by Legionella pneumophila and its role in bacterial ecology and pathogenesis. Appl Environ Microbiol 1998, 64:3127-3133.
3.
Horwitz MA, Silverstein SC: Legionnaires’ disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J Clin Invest 1980, 66:441-450.
4.
Horwitz MA: The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med 1983, 158:2108-2126.
5.
Horwitz MA: Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. J Exp Med 1987, 166:1310-1328.
6.
Horwitz MA: Phagocytosis of the Legionnaires’ disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil. Cell 1984, 36:27-33.
7.
Payne NR, Horwitz MA: Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med 1987, 166:1377-1389.
8.
Horwitz MA, Silverstein SC: Interaction of the legionnaires’ disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication. J Exp Med 1981, 153:398-406.
9.
Horwitz MA, Maxfield FR: Legionella pneumophila inhibits acidification of its phagosome in human monocytes. J Cell Biol 1984, 99:1936-1943.
10. Horwitz MA: Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med 1983, 158:1319-1331. 11. Swanson MS, Isberg RR: Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun 1995, 63:3609-3620. 12. Bozue JA, Johnson W: Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion. Infect Immun 1996, 64:668-673. 13. Abu Kwaik Y: The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 1996, 62:2022-2028. 14. Venkataraman C, Haack BJ, Bondada S, Abu Kwaik Y: Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires’ disease bacterium. J Exp Med 1997, 186:537-547. 15. Harb OS, Venkataraman C, Haack BJ, Gao LY, Kwaik YA: Heterogeneity in the attachment and uptake mechanisms of the Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. Appl Environ Microbiol 1998, 64:126-132. 16. Dreyfus LA: Virulence associated ingestion of Legionella pneumophila by HeLa cells. Microb Pathog 1987, 3:45-52. 17.
Cianciotto NP, Stamos JK, Kamp DW: Infectivity of Legionella pneumophila mip mutant for alveolar epithelial cells. Curr Microbiol 1995, 30:247-250.
18. Garduno RA, Quinn FD, Hoffman PS: HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila. Can J Microbiol 1998, 44:430-440. 19. Rowbotham TJ: Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci 1986, 22:678-689. 20. Cirillo JD, Falkow S, Tompkins LS: Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect Immun 1994, 62:3254-3261. 21. Byrne B, Swanson MS: Expression of Legionella pneumophila • virulence traits in response to growth conditions. Infect Immun 1998, 66:3029-3034. This paper describes various conditions that allow enhanced growth within cultured macrophages.
MC2111.QXD
12/14/1999 10:44 AM
34
Page 34
Host–microbe interactions: bacteria
22. Brieland JK, Fantone JC, Remick DG, LeGendre M, McClain M, • Engleberg NC: The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire’s disease. Infect Immun 1997, 65:5330-5333. This paper describes potential phenotypic switch involving virulence traits as a function of growth conditions. 23. McDade JE, Shepard CC: Virulent to avirulent conversion of Legionnaires’ disease bacterium (Legionella pneumophila) — its effect on isolation techniques. J Infect Dis 1979, 139:707-711. 24. Catrenich CE, Johnson W: Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium. Infect Immun 1989, 57:1862-1864.
38. Vogel JP, Andrews HL, Wong SK, Isberg RR: Conjugative transfer •• by the virulence system of Legionella pneumophila. Science 1998, 279:873-876. This paper, along with [37••] shows that the dot/icm genes code for a type IV secretion system used to alter the endocytic pathway of macrophages. 39. Winans SC, Burns DL, Christie PJ: Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol 1996, 4:64-68. 40. Roy CR, Berger KH, Isberg RR: Legionella pneumophila DotA • protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol Microbiol 1998, 28:663-674. See annotation for [41•].
25. Marra A, Blander SJ, Horwitz MA, Shuman HA: Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc Natl Acad Sci USA 1992, 89:9607-9611.
41. Wiater LA, Dunn K, Maxfield FR, Shuman HA: Early events in • phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect Immun 1998, 66:4450-60. This paper and [40•] provide evidence that the dot/icm genes function early during infection of macrophages.
26. Brand BC, Sadosky AB, Shuman HA: The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages. Mol Microbiol 1994, 14:797-808.
42. Husmann LK, Johnson W: Cytotoxicity of extracellular Legionella pneumophila. Infect Immun 1994, 62:2111-2114.
27.
Berger KH, Isberg RR: Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 1993, 7:7-19.
28. Berger KH, Merriam JJ, Isberg RR: Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol 1994, 14:809-822. 29. Roy CR, Isberg RR: Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages. Infect Immun 1997, 65:571-578. 30. Sadosky AB, Wiater LA, Shuman HA: Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect Immun 1993, 61:5361-5373. 31. Purcell M, Shuman HA: The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages. Infect Immun 1998, 66:2245-2255. 32. Segal G, Shuman HA: Characterization of a new region required for macrophage killing by Legionella pneumophila. Infect Immun 1997, 65:5057-5066.
43. Kirby JE, Vogel JP, Andrews HL, Isberg RR: Evidence for pore • forming ability by Legionella pneumophila. Mol Microbiol 1998, 27:323-336. The authors describe the contact-dependent cytotoxicity caused by the dot/icm genes. 44. Elliott JA, Johnson W: Virulence conversion of Legionella pneumophila serogroup 1 by passage in guinea pigs and embryonated eggs. Infect Immun 1982, 35:943-946. 45. Pruckler JM, Benson RF, Moyenuddin M, Martin WT, Fields BS: Association of flagellum expression and intracellular growth of Legionella pneumophila. Infect Immun 1995, 63:4928-4932. 46. Merriam JJ, Mathur R, Maxfield-Boumil R, Isberg RR: Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect Immun 1997, 65:2497-2501. 47.
Cianciotto NP, Eisenstein BI, Mody CH, Toews GB, Engleberg NC: A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect Immun 1989, 57:1255-1262.
33. Andrews HL, Vogel JP, Isberg RR: Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect Immun 1998, 66:950-958.
48. Szeto L, Shuman HA: The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect Immun 1990, 58:2585-2592.
34. Gao LY, Harb OS, Abu Kwaik Y: Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant host cells, mammalian macrophages and protozoa. Infect Immun 1997, 65:4738-4746.
49. Quinn FD, Tompkins LS: Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities. Mol Microbiol 1989, 3:797-805.
35. Swanson MS, Isberg RR: Identification of Legionella pneumophila mutants that have aberrant intracellular fates. Infect Immun 1996, 64:2585-2594.
50. Rdest U, Wintermeyer E, Ludwig B, Hacker J: Legiolysin, a new hemolysin from L. pneumophila. Zentralbl Bakteriol 1991, 274:471-474.
36. Vogel JP, Roy C, Isberg RR: Use of salt to isolate Legionella pneumophila mutants unable to replicate in macrophages. Ann NY Acad Sci 1996, 797:271-272.
51. Liles MR, Viswanathan VK, Cianciotto NP: Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect Immun 1998, 66:1776-1782.
37. ••
Segal G, Purcell M, Shuman HA: Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci USA 1998, 95:1669-1674. See annotation for [38••].
52. Stone BJ, Abu Kwaik Y: Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect Immun 1998, 66:1768-1775.