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A. Marra Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachussetts 02139

H. A. Shuman Department of Microbiology, Columbia Uni ve rsity College of Physicians and Surgeons, New York, NY 10032 KEY WORDS: proteolysis, cytotoxins, antigenicity, immune response, hemolytic activity

CONTENTS . . . .

52 52 54 55

GENETICS IN L. PNEUMOPHlLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


APPROACHES TO IDENTIFYING GENES FOR VIRULENCE FACTORS Cloning by Functiona l A ssay . . .. . .. . .. . . . . . . . . . . . .. . .. . Cloning Immunodominant Antigens . . . . . . . . . . . . .. . . .. . . . . . Complementation of A virulent Mutants of L. pneumophila . . . .... . Other Factors A ffecting Virulence . . . . . . . . . . . . . . . . . . . . . . . .

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57 58 59 61 63

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INTRODUCTION TO LEGIONELLA PNEUMOP HILA Cell B iology . . . . . . . . . . . . . . . . . . . . . . . . Host Range . . . . . . . . . . . . . . . . . . . . . . . . . Virulence Conversion . . . . . . . . . . . . . . . . . . .



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Every intracellular pathogen must have evolved mechanisms by which it gains access to its host, evades extracellular and intracellular host defenses, replicates within the confines of a cell, and spreads to other cells and hosts. Certain strategies at each step may be shared among pathogens, yet the entire program is unique for a given pathogen. Cell biology can reveal these strategies to us, but genetics enable us to define the molecular bases for these events. In a relatively short period following the discovery of Legionella pneumophila in 1976, we have learned in exquisite detail how this organism infects its host cell, subverting the cell's defense machinery as it is destroyed. 51




Despite great effort by several laboratories, genetic analysis of L. pneumophila has lagged behind the cell biology, mainly because new tools and techniques were needed with which to study this organism. Only recently have genetic strategies, in vitro models, and molecular cloning come together to identify and clone factors of direct relevance in pathogenesis.

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INTRODUCTION TO LEGIONELLA PNEUMOPHILA Legionella pneumophila, the gram-negative bacterium responsible for Legion­ naires' disease and the less severe Pontiac fever, is a facultative intracellular pathogen ubiquitous in nature. L. pneumophila is well-suited to laboratory study as it can be grown in artificial rich as well as semidefined media, and in a wide range of host cells, including mammalian cells and protozoa. The doubling time of L. pneumophila in rich medium is approximately two hours, similar to that observed in human peripheral blood monocytes,the best-studied hosts for L. pneumophila.

Cell Biology Since its isolation, much has been learned about the cell biology of L. pneumophila and how its exerts its pathogenic effects. The work of several groups, notably Horwitz et al (5,8, 17,52-54, 5(H}1, 85), have elucidated in detail the pathway of this organism through human macrophages. Figure 1 outlines this pathway and the genes of L. pneumophila that are or may be involved at each step. This figure also notes the steps in this pathway which L. pneumophila shares with other human intracellular pathogens. L. pneu­ mophila attaches to its host cell via complement receptor CR3,and, to a lesser extent, to CRl (4, 85). Binding of CRI and CR3 occurs through complement component C3 fixation on the bacterial surface (4, 5); the ligand for C3 on L. pneumophila is the major outer membrane protein (MOMP), a 28-kd porin (4,46). In addition to L. pneumophila, Mycobacterium tuberculosis (84), M. leprae (93), Leishmania donovani (10), L. major (80, 88), and Histoplasma capsulatum (15) utilize complement receptors on phagocytes. Following attachment, L. pneumophila is taken up by cells through an unusual mechanism termed coiling phagocytosis, in which a single pseudopod extends outward from the cell surface and coils around the bacterium (54); this mechanism of uptake has also been observed for L. donovani (23). If the bacteria are first opsonized with anti-L. pneumophila antibodies, the bacteria enter the cells by conventional phagocytosis (54), suggesting an alternate means of entry into cells lacking surface CR3 molecules, as might occur in the aquatic environment. Inside the cell, L. pneumophila resides within a specialized phagosome which does not acidify, and apparently recruits host cell smooth vesicles, mitochondria. and ribosomes (52,56). The bacteria prevent or fail to stimulate





Physical Features

Cell surtace

CR1. CR3



Does not aCidify

- Recruits cellular organelles •

Nutritional requirements




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lysosome fusion

Known or possible t. pneumophlls genes Involvea Other intracellular pathogens

Figure 1




icm (dot)

msp? (pro?)




LPS? Mycobacterium tuberculosis M.leprae Leischman;a major L. donovani

L donovan;

M. tuberculosis

M. micro'; Toxoplasma gondii Chlamydia psiUaci (inhibit fusion only)

Pathway of L. pneumophila infection of human macrophages. Headings on the left

correspond to the stages defined in the top line, describing the physical features observed at each stage, known or potential genes of L. pneumophila involved, and human intracellular pathogens that utilize the indicated strategies (l, 10, 23, 45, 63, 64, 70, 80, 88, 93). A question mark after a gene indicates that its role in virulence is unknown.

phagosome-lysosome fusion, and multiply within this phagosome (53, 57). Other pathogens that inhibit phagosome-lysosome fusion as part of their intracellular lifestyle are M. tuberculosis (1) , M. microti (70) , Chlamydia psittaci (45), and Toxoplasma gondii (64). The host cell eventually becomes filled with bacteria and lyses. Although cytotoxic activity has been reported in L. pneumophila (44, 66, 89), it is not clear whether it is the action of a specific cytotoxin or simply the large numbers of intracellular bacteria that lyse the cell. The infection cycle is repeated as extracellular L. pneumophila infect new host cells. In mammals, the host limits infection by L. pneumophila predominantly by a cell�mediated response. Although there is a strong humoral response during infection, antibodies provide little defense and may in fact promote the uptake of L. pneumophila (60), Macrophages activated by lymphokines are no longer good hosts for L. pneumophila, as they limit infection in two ways: ftrst, by decreasing phagocytosis of L. pneumophila, possibly through decreased expression of CRl and CR3 (37, 58); and second, by decreasing intracellular iron pools by down-regulation of their transferrin receptors (17) . Iron is required for both intra- and extracellular growth of L. pneumophila (16, 17, 91); compounds that chelate intracellular iron or otherwise deplete the intracellular iron pool inhibit replication of L. pneumophila (17, 18). There





is currently much interest in understanding the mechanism of iron uptake in L. pneumophila. These bacteria do not produce detectable siderophores (92),

but a periplasmic iron reductase activity has been reported (62). As many pathogens require iron for intracellular growth (99), elucidation of the iron-acquiring capabilities of L. pneumophila may help us to identify novel mechanisms for iron acquisition.

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Host Range L. pneumophila is ubiquitous in nature, living in aquatic environments usually in association with freshwater amoebae. L. pneumophila can infect and replicate in several unicellular hosts, including Acanthamoeba castellanii Neff (51 ), A. royreba (98), H artmanella vermiformis (40, 68), Naegleriajowleri (81 ), N. lovaniensis (98), and Tetrahymena pyriformis (41, 42). In addition, L. pneumophila can utilize the extracellular products of blue-green algae for extracellular growth (96). These findings may explain the widespread distri­ bution of L. pneumophila in both natural and man-made water systems, the latter being the primary source of its spread to humans. The animal model for Legionnaires' disease is the guinea pig, which is highly susceptible to infection from L. pneumophila and, following aerosol challenge, exhibits a fatal pneumonia very similar to that seen in human infections (3, 7, 31, 32, 65). L. pneumophila is capable of infecting and replicating within several different mammalian cell types at varying efficiencies; its primary host cells are human peripheral blood monocytes and alveolar macrophages (57). L. pneumophila can also grow in the cell lines MRC-5 (human embryonic lung fibroblasts) (30, 82, 101), Vero (African Green monkey kidney) (82), Hep-2 (human epithelial laryngeal carcinoma) (30, 82), U937 (human histiocytic lymphoma) (86), HL-60 (human leukemia) (73), and HeLa (human cervical carcinoma) (30) cells. Mice are highly resistant to infection from L. pneumophila and most mouse peritoneal macrophages are nonpermissive for growth of this pathogen, including BDFl, DBA/2, C3H/HeN, C57BLl6, and BALB/c (102). However, one inbred mouse strain, A/J, is able to support intracellular growth of virulent L. pneumophila (02) , and L. pneumophila has been reported to form plaques on mouse L929 cells (39). The fact that after infection with L. pneumophila, bacteria are found only in the lungs and secondarily in the spleen makes it difficult to evaluate the physiological relevance, if any, of L. pneumophila interactions with other cell types in vivo. The most common cell lines used to study L. pneumophila are HL-60 and U937, although an amoebae model has recently been reported (79). Assays for L. pneumophila virulence are those that quantitate intracellular growth (57), mammalian cell killing (cytotoxicity assays using the dye MTT) (73), or both (plaque assays that depend on both cell killing and intracellular growth) (7 2a).



Virulence Conversion An interesting phenomenon is the irreversible change in L. pneumophila from virulent to avirulent. This conversion was observed as early as 1979 (75), but the genetic events leading to avirulence are still not understood. McDade & Shepard (75) reported that L. pneumophila repeatedly cultivated for 6 months on Mueller-Hinton agar supplemented with 1 % hemoglobin and 2% IsoVitalex

�onated eggs of > 107 colony-forming units (CFU) and

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had an LD50 for emb

for guinea pigs of 10 CFU, whereas L. pneumophila propagated in either guin ea pigs or in embryonated eggs had LD50 values of < 10 CFU for both. The authors also noted that L. pneumophila virulence was lost after only 10 passages on supplemented MUeller-Hinton agar, and that bacteria from infected guinea pigs could be cultivated on another medium, Feeley-Gorman (ABCYE) agar (38), but not on supplemented MUeller-Hinton agar.

It became apparent that supplemented Mueller-Hinton agar actually inhib­ ited the growth of virulent L. pneumophila, which could in fact be recovered

from these plates to grow on ABCYE agar, the current medium of choice for L. pneumophila. The inhibitory factor was identified as NaCl, at a concen­ tration of 0. 65% (20). Work from our laboratory has shown that avirulent organisms can easily be isolated from ABCYE agar containing 0.65% NaCI (A. Marra, J. Rogers, unpublished results). More work is needed to elucidate the genetic mechanism governing this conversion and to determine whether all avirulent isolates obtained in this way fall into the same class with respect to their block in the infection pathway.

GENETICS IN L. PNEUMOPHILA Many investigators have focused on the genetic manipulation as well as the

cell biology of


pneumophila. Methods of introducing DNA into this

organism are limited to conjugation and electroporation. Transduction has not been possible without the isolation of a bacteriophage t hat infects L.

pneumophila. Conjugation between Escherichia coli and L. pneumophila was initially hampered by the poor mating frequencies observed for L. pneu­ mophila at 10-7 to 10-8 per recipient (24, 74, 78). Restriction mutants of L. pneumophila have been isolated that increase this frequency to 10-3 to 10-1 per rec ipien t (28, 74), and these mutants have greatly facilitated genetic experiments. Recently, electroporation has been used successfully to introduce IncP and IncQ plasmids as well as pUC8 derivatives into L. pneumophila.


This occurs at frequencies of approximately 10 colonies per f.Lg of plasmid DNA in a restriction mutant of strain Philadelphia-l (M. Bouchard & A. Marra, unpublished observations). Only a few groups have reported using transposon mutagenesis with any

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success in L. pneumophila, but recent results are encouraging. Keen et al (67) introduced Tn5 into L. pneumophila using pRK340, a temperature-sensitive derivative of plasmid RK2, by screening for kanamycin-resistant mutants at the nonpermissive temperature; however, this occurred at frequencies too low to be useful in mutagenesis experiments. Attempts to deliver Tn phoA into L. pneumophila via a suicide vector system based on RP4 mobilization functions and R6K replication functions have also been unsuccessful (94; A. Marra, unpublished results). Due to the low transposition frequency of Tn5 in this bacterium, all kanamycin-resistant mutants were actually the result of chro­ mosome mobilization of the donor strain of E. coli via an integrated RP4-2-Tc::Mu supplying mobilization functions. All these attempts to intro­ duce transposons into the chromosome of L. pneumophila underline the importance of a reliable suicide vector system for this organism. To circumvent this problem, an indirect method of TnphoA mutagenesis has been proposed (N. C. Engleberg, personal communication) in which a L. pneu­ mophila library in E. coli is mutagenized and PhoA + cosmids introduced by homologous recombination onto the chromosome of L. pneumophila. This approach is limited by its dependence on L. pneumophila gene expression in E. coli. With a similar aim, a Tn903 derivative carrying a truncated lacZ gene, Tn903dIIlacZ, has been shown to create translational gene fusions in L. pneumophila when introduced on a ColEl derivative that is rapidly lost in the absence of selection (A. Sadosky & L. Wiater, unpublished observations). Bacteriophage Mu also transposes in L. pneumophila (78), and although prophage induction is lethal, Mu is unable to complete its life cycle to form active phage particles in L. pneumophila. Taking advantage of this, Engleberg & co-workers (manuscript submitted) have developed the transposon Mud phoA to generate fusions to genes encoding exported proteins. MudphoA was constructed by cloning the phoA gene from E. coli and the kanamycin­ resistance gene from Tn5 into MudlI404 l (19), which contains the tempera­ ture-sensitive repressor allele, cts. The cts gene may represent one drawback to using Mud phoA to isolate avirulent mutants, because although growth of MudphoA-containing L. pneumophila at 37°C results in no apparent derepres­ sion of Mu, growth at higher temperatures, such as may be observed in febrile animals, may cause Mu transposition and decreased viability of the mutants of L. pneumophila in vivo. Mudp hoA Tn903dlIlacZ, and the indirect method of creating phoA fusions discussed above have generated avirulent derivatives of L. pneumophila (see below). Using well-defined mutants of E. co li several investigators have cloned functional homologs of L. pneumophila. The recA gene from L. pneumophila was cloned and the protein found to promote homologous recombination and increase survival of E. coli HBlOl (recA) after UV exposure ( 3 3 ; L. Szeto, unpublished observations). RecA of L. pneumophila is 37.5 kd and is ,


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antigenically related to RecA of E. coli. One researcher detected a four- to fivefold induction of L. pneumophila RecA after UV irradiation but no induction of a lambda prophage (33). However, independent studies have shown no induction of L. pneumophila RecA following mitomycin C or UV treatment when analyzed by Western blot or by using a strain carrying a L. pneumophila recA-lacZ fusion (L. Szeto, unpublished observations). In addition, a 20- to 50-fold increase in lambda induction was observed in an E. coli recA(A +) lysogen carrying L. pneumophila recA on a low copy plasmid (L. Szeto, unpublished observations). These results demonstrate that the L. pneumophila RecA protein may partially compensate for most E. coli RecA activities, althqugh no homology at the DNA level was observed by Southern analysis. A IlrecA strain of L. pneumophila is still able to grow within and kill differentiated HL-60 cells (R. Bryan, L. Wiater, & L. Szeto, unpublished observations). As a second example of this approach, our laboratory has cloned a trpE homolog from L. pneumophila that complements a deletion mutant of E. coli (L. Szeto, A. Marra, unpublished observations). Although these genes may have no role in virulence, they demonstrate the validity of this approach. Endogenous plasmids have been found in at least two serogroups of L. pneumophila, but have no role in virulence. An early report (69) described the isolation of a 30-MDa plasmid from L. pneumophila serogroup 2; this plasmid was cured spontaneously after repeated subculturing on ABCYE agar. The authors observed no change in the virulence of the cured strains for mice. In another study, a 128-kb plasmid of L. pneumophila serogroup 1, pCHl , could be conjugally transferred to other strains of L. pneumophila serogroup 1 but not to serogroup 3 strains or to E. coli (c. S. Mintz, personal communication). When pCRI was introduced into avirulent or virulent serogroup 1 strains that had been lacking this plasmid, no change in their abilities to grow within either U937 cells or in H. vermiformis could be detected. These results confirm those of the earlier report, suggesting that genes required for virulence are chromosomally located in L. pneumophila. APPROACHES TO IDENTIFYING GENES FOR VIRULENCE FACTORS

Many groups have undertaken genetic approaches to understand the pathogenesis of L. pneumophila. Three distinct approaches have been used: cloning genes of L. pneumophila by functional assay; cloning immunodomin­ ant antigens of L. pneumophila; and cloning genes of L. pneumophila that complement mutants of L. pneumophila. These studies have provided some insight into the genes involved in pathogenesis; however, many more important advances have been made in understanding this organism, in a way "taming" a bacterium from the wild so that it can be manipulated in the laboratory.





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As this review focuses on the impact of genetics on understanding L. pneumophila virulence, genes that do not play a role in pathogenesis are not discussed in detail. However, these experiments are mentioned to illustrate the genetic approaches taken with this organism and the successes and pitfalls that have been encountered. These efforts have laid the foundation for future research using tools and techniques specifically designed for L. pneumophila. The remainder of this review discusses genetic loci of known and potential virulence factors in the context of the different approaches mentioned above. Cloning by Functional Assay L. pneumophila has been observed to produce extracellular proteolytic, cytotoxic, and hemolytic activities (44,66, 89,90,95, 100). There has been much interest in these activities and their potential roles as virulence factors. For this reason and perhaps due to the relative ease with which genes encoding these activities can be isolated, several laboratories have set out to clone these genes. However, with these examples comes a cautionary note: not everything secreted, antigenic, or homologous to a virulence factor is, in fact, involved in virulence. The gene for the major secretory protein (called msp or proA; we refer to this gene as msp) was cloned by two groups independently (90, 95), after reports of the antigenicity and tissue-destroying properties of this molecule. Several lines of experimentation suggest that Msp may play an important role in virulence. This protein, a 38-kd Zn2+ metalloprotease, has been reported to be the most abundant protein in culture supernatants of L. pneumophila, with proteolytic activity against casein, collagen, gelatin, and bovine insulin (9); hemolytic activity has also been reported for this protein (66). In addition, there is some evidence that Msp is cytotoxic for a variety of cell types, including McCoy, HeLa,CHO,human embryonic lung fibroblasts,and Green monkey kidney cells (9), and aerosolized preparations of purified protease causes lesions in guinea pig lungs very similar to those induced by L. pneumophila itself (2, 29). It has recently been demonstrated that Msp is produced intracellularly, starting at 1 2 hr and increasing up to 48 hr postinfection,and is localized to L. pneumophila-containing phagosomes and to the plasma membrane (D. L. Clemens & M. A. Horwitz, personal communication). Lastly, it has been shown that guinea pigs infected with L. pneumophila develop a strong cell-mediated immune response to Msp, and that vaccination with Msp not only induces cell-mediated and humoral immune responses, but results in protective immunity when these animals are later challenged with L. pneumophila (12). The msp gene of L. pneumophila was cloned after screening L. pneumophila libraries in E. coli for clones capable of hydrolyzing casein. The gene has been sequenced and found to encode a

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putative signal sequence and to have homology to elastase of Pseudomonas aeruginosa (9), as well as to a lecithinase of Listeria monocytogenes (76); both of these have been implicated in virulence. Southern hybridization experiments have revealed msp homologs in all strains of L. pneumophila examined, though not in other Legionella species (89). An msp::Tn9 mutant constructed by allelic exchange shows less than 0. 1 % of the proteolytic activity of the wild-type strain, indicating that this protein is responsible for essentially all of the extracellular proteolytic activity of L. pneumophila (95). This mutant is capable of intracellular growth and cell killing in an HL-60 cell model of infection by L. pneumophila. and is as virulent as its isogenic parent strain for guinea pigs (14). Interestingly, both Msp-I' and Msp L. pneumophila cause similar lesions in the guinea pig lungs, suggesting that another factor must be involved in this phenomenon (14). These results clearly demonstrate that a molecule that is not a virulence factor can cause protective immunity, and may present an opportunity for the host immune system to recognize an intracellular pathogen. Another extracellular enzyme has recently been cloned from L. pneu­ mophila, and although its role in pathogenesis has not been identified as yet, this enzyme has hemolytic activity on human, sheep, and canine erythrocytes and appears to be responsible for formation of brown pigment in cultures of L. pneumophila. The gene encoding this enzyme has been cloned from a L. pneumophila library in E. coli that had been screened on canine blood agar plates for hemolytic activity, and has been designated lly, for legiolysin (100). Only strains of L. pneumophila exhibit homology to the lly gene and cross-react with antibodies against this 39-kd protein. This gene is distinct from msp, and does not hybridize to any other gram-negative pathogens examined (6). It is not clear what role, if any, Lly may play in pathogenesis, as it has no proteolytic or cytotoxic activity, although independent experiments may address this question. Recently, a mutant of L. pneumophila has been isolated after Tn903dIIlacZ mutagenesis that does not produce brown pigment and shows no attenuation in its ability to kill differentiated HL-60 cells (A. Sadosky, unpublished observations). It is not yet known whether the Tn903dIIlacZ insertion is in the fly gene in this mutant. -

Cloning Immunodominant Antigens Another common approach to cloning genes from L. pneumophila also relies on their expression in E. coli, screening with antibodies directed against either purified antigens of L. pneumophila or whole bacteria. In one of the earliest reports on L. pneumophila genetics, Engleberg et al (36) showed that antigens of L. pneumophila could be cloned, expressed, and correctly localized in E. coli, and from this starting point several groups have cloned and characterized antigenic molecules from L. pneumophila. Here again, the aim was to identify

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potential virulence factors. Although this approach depends for success on the proper expression of antigens of L. pneumophila in E. coli, several interesting genes have been isolated. Probably one of the first genes cloned by this method, and consequently the best-studied, is mip (27). This gene encodes a 24-kd surface antigen that was isolated after screening a clone bank from L. pneumophila with anti-L. pneumophila antiserum. The Mip protein is highly basic, and optimizes L. pneumophila virulence (macrophage infectivity potentiator) (27, 34). A mip· mutant was constructed by directed allelic exchange and found to grow intracellularly in the cell line U937 (86) and in alveolar macrophages with the same kinetics as the wild-type strain; however, l O-fold fewer bacteria were recovered at early times postinfection unless inoculum 80-fold larger in size was used (27). This mutant also exhibits an -IOO-foid increase in lethal dose in guinea pigs compared to the wild-type strain (26). The DNA seql;lence of mip reveals homologies to a 27-kd surface protein of Chlamydia trachomatis (72) and to human proteins and proteins of Neurospora crassa that bind the immunosuppressive drug FK506 and have prolyl isomerase activity (97). Conflicting reports have shown Mip not to retain this activity (N. C. Engleberg, unpublished results), or to have prolyl isomerase activity inhibited by FK506 (G. Fischer, et aI, submitted for publication). One group found that Mip inhibits protein kinase C from human neutrophils (N. C. Engleberg, unpublished results). The discovery of protein kinase C inhibition by Mip has led to the suggestion that Mip may interfere with early intracellular bactericidal mechanisms that depend on protein kinase C. All isolates of L. pneumophila examined show hybridization to a mip gene probe as well as cross-reactivity with anti-Mip antibodies; weaker reactivities were observed with other Legionella species, indicating the presence of mip homologs throughout the genus (25). Another gene cloned as a major antigen of L. pneumophila is htsB. Several results suggest that this 60-kd protein may be a heat shock factor in L. pneumophila. HtsB is localized to the periplasm of L. pneumophila, and its expression is increased twofold following heat shock (47, 48). DNA sequence analysis revealed an E. coli consensus heat shock promoter upstream of htsA, the first gene in the htsAB operon; temperature-dependent transcription of htsB in E. coli appears to require the rpoH gene (encoding the heat shock sigma factor, sigma-32) (47). In addition, HtsB has 85% homology with E. coli GroEL and Coxiella burnetii HtpB, and 76% homology with a 65-kd heat shock protein of Mycobacterium tuberculosis, all at the amino acid level (48). However, despite common epitopes, htsB of L. pneumophila is not able to complement either a groES or a groEL mutant of E. coli (47). HtsB is often referred to as a genus-common antigen, with epitopes specific to Legionella

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(87), and may show promise as a diagnostic tool for L. pneumophila, although the role in virulence of htsB has not yet been defined. Using this same approach, the gene for a 19-kd peptidoglycan-associated protein has been cloned. This outer membrane protein, known as Ppl (71) or Pal (35), shows homology to E. coli and peptidoglyc an-associated lipoproteins of Haemophilus influenzae. DNA sequence comparison of the genes of E. coli, H. influenza, and L. pneumophila reveal s a signa l peptidase II consensus signal sequence (35), consistent with the observation that the L. pneumophila Pal is also a lipoprotein. The gene for the major outer membrane protein (MOMP) has defied cloning by this method. The most abundant protein produced by L. pneumophila, MOMP is a cation-specific porin and binds complement components C3 and C3bi, and so plays a key role in the uptake of the bacteria by CR3+ phagocytes (4, 46). The gene for MOMP, ompS, has recently been cloned using a DNA probe based on the N-terminal amino acid sequence of purified MOMP (49). It has long been believed that MOMP is a single protein species of 28 kd; recent work has demonstrated the existence of two identical subunits, one of which (31 kd) is covalently attached to peptidoglycan (50). These proteins are synthesized from one gene encoding a single transcript, and mature MOMP is a multimer of these subunits, with a molecular weight of at least 100 kd (49,50). With the ompS gene in hand, it should be possible to firmly establish the role of MOMP during infection. Although the antigenic approach described in this section has yielded at least one virulence-associated factor, it is not clear whether all such genes will prove to be as interesting as mip. This biochemical approach has definite limitations, as it is dependent on the correct expression and localization of proteins in E. coli; however, only under conditions approaching those in vivo can we begin to ask specific questions about virulence. Complementation of Avirulent Mutants of L. pneumophila The most straightforward approach to address the question of virulence directly is to develop methods to isolate and complement avirulent derivatives of L. pneumophila. There has been a recent surge in this direction, as more and more investigators develop sophisticated strategies to isolate particular mutants of L. pneumophila. Using some of the genetic tools described above, several groups have isolated avirulent derivatives of L. pneumophila. For example, preliminary experiments using the indirect TnphoA mutagenesis strategy have identified three out of 39 PhoA + strains of L. pneumophila with decreased virulence in the U937 cell model (N. C. Engleberg, personal communication). MudphoA mutagenesis has also generated at least one mutant that is unable to grow in

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U937 cells (N. C. Engleberg & coworkers, submitted). By creating transla­ tional fusions to E. coli lacZ using Tn903dIIlacZ mutagenesis, approximately 80 out of 4000 kanamycin-resistant, Lac + mutants are attenuated in their abilities to kill macrophages derived from the HL-60 cell line (A. Sadosky & L. Wiater, unpublished observations). These studies should facilitate the isolation of different classes of mutants of L. pneumophila. One class of mutants (see section on virulence conversion) is represented by mutant 25D (55). The intracellular pathway of this avirulent derivative of L. pneumophila Philadelphia- l is described in Figure 2. Mutant 25D was isolated after 13 serial passages on supplemented Mueller-Hinton agar, and so is resistant to concentrations of NaCl that are inhibitory to Philadelphia-l growth. Detailed examination of this mutant's intracellular pathway revealed an inability to inhibit phagosome-lysosome fusion; in addition, phagosomes containing 25D acidify and do not recruit organelles (55). As a consequence, 25D is unable to grow intracellularly within, or kill, any cell type examined, and does not cause disease in guinea pigs (11). An interesting aspect of 25D's intracellular profile is its ability to survive within the phagolysosome up to 24 hours after fusion has occurred (55). This suggests that L. pneumophila is resistant to lysosomal degradation but is unable to replicate in this harsh environment.

Figure 2

Comparison of the intracellular pathways of wild-type L. pn eumophila and mutant

25D. Both strains bind and enter monocytes in the same way, but whereas the wild-type inhibits phagosome-lysosome fusion and phagosome acidification as it recruits cellular organelles, 25D is incapable of these activities but remains viable within the phagolysosome for up to 24 hours (Reprinted from ref. 55 with permission).

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The inability of 25D to grow within and kill cells was complemented after introduction of a wild-type genomic library on the cosmid vector pLAFRl (43) and selection for clones capable of forming plaques on an HL-60 cell monolayer. The complementing DNA, referred to as the icm locus (for intracellular multiplication) confers on 25D the ability to grow within and kill HL-60 cells, in addition to the ability to cause lethal pneumonia in guinea pigs at LDso values comparable to wild-type. Interestingly, this locus does not render 25D NaCI-sensitive, indicating that this phenotype is due either to a dominant mutation or to a mutation outside the cloned region (72a). In independent experiments,complementation of avirulent mutants whose intracellular fates are similar to that of 25D has implicated the same region of Philadelphia- l DNA (K. Berger & R. Isberg,personal communication). These mutants were isolated following transposon mutagenesis of a Thy" mutant of L. pneumophila (see below) and an intracellular enrichment scheme that relied on the idea that avirulent mutants would be unable to grow intracellularly and therefore would not undergo thymine1ess death. The complementing DNA was called the dot locus, for direction of organelle trafficking (K. Berger & R. Isberg, personal communication). This same locus has been implicated in virulence in yet another set of independent experiments. In screening some 4000 mutants of Tn903dIIlacZ mutagenesis, one avirulent class was found to contain transposon insertions in this region (A. Sadosky & L. Wiater, personal communication). It is not yet known how this region of L. pneumophila DNA is involved in virulence, although derivatives of 25D carrying the icm (dot) locus inhibit phagosome-lysosome fusion and recruit organelles. Other Factors Affecting Virulence Several other factors have been suggested to have roles in virulence. Some, such as iron (discussed above) and auxotrophy, are not virulence factors per se, but do affect the ability of L. pneumophila to grow intracellularly. Certain auxotrophic requirements affect the intracellular growth capabilities of L. pneumophila. Tryptophan and thymidine auxotrophs of L. pneumophila have been described, and although tryptophan auxotrophs can survive and grow intracellularly, thymidine auxotrophs cannot grow intracellularly (77). This effect of thymidine auxotrophy may be due either to these mutants undergoing thymineless death, or to their inability to grow in the absence of thymidine and thus to inhibit phagosome-lysosome fusion (77). Addition of thymidine to the tissue culture medium restores to these mutants the ability to grow intracellularly. These studies demonstrate the dependence of intracellular pathogens on the metabolic activities of their hosts. Other possible factors involved in virulence reside on the surface of L. pneumophila. These include lipopolysaccharide (LPS), flagella, and serum resistance. Membranes of L. pneumophila that do not contain Msp induce

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cell-mediated and protective immunity in vaccinated guinea pigs (1 3). To date, one LPS mutant of L. pneumophila has been described (C. S. Mintz, personal communication). This mutant does not bind a monoclonal antibody directed against L. pneumophila serogroup 1, yet it has no obvious differences in its LPS compared with wild-type. This subtle change in LPS has no effect on the mutant's ability to grow in U937 cells or in Hartmanella vermiformis (C. S. Mintz, personal communication). Flagella have been observed on L. pneumophila (21, 22, 83), and a recent report has identified a 47-kd flagellin subunit whose expression appears to be regulated by temperature (83). A number of nonmotile mutants with severely decreased abilities to grow in and kill differentiated HL-60 cells have been derived independently (R. Bryan, unpublished observations). Some evidence suggests that serum resistance may be a requisite for virulence. L. pneumophila is highly resistant to the bactericidal effects of serum (59), with no loss in the number of colony-forming units after incubation for one hour in the presence of 50% serum. This resistance is also observed when L. pneumophila is incubated with serum in the presence of anti-L. pneumophila antibody (59). One mutant isolated in our laboratory is more serum-sensitive than its wild-type parent, and shows attenuation in its ability to grow within human monocytes. Although it has not been shown that the two phenotypes are linked, it is not unreasonable to assume that a decrease in serum resistance in vitro would result in decreased ability to survive serum concentrations encountered in culture media or in vivo. CONCLUSIONS

Since the first major outbreak of Legionnaires' disease in 1 976 and the subsequent identification of L. pneumophila, we have learned much about both the cell biology and the genetics of this organism. Limitations on the genetic manipulations possible in L. pneumophila have forced the develop­ ment of techniques directly for L. pneumophila. In the near future, we can expect to have molecular evidence for the cell biological events observed during infection. Because features of its infection pathway are shared with other pathogens,the entry,survival, and growth strategies of L. pneumophila may help in the understanding of other parasitic diseases whose agents are not so easily studied as L. pneumophila. ACKNOWLEDGMENTS

Studies from our laboratory were supported by grant AI23549 from the National Institutes of Health. H. A. S. is supported by a Faculty Research Award from the American Cancer Society. We thank our colleagues for their cooperation in providing unpublished information.



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Genetics of Legionella pneumophila virulence.

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