Secretion and Export in Legionella

Secretion and Export in Legionella

Nicholas P. Cianciotto

Abstract

L. pneumophila secretes many factors that promote its growth and persistence within the environment, various types of host cells, and the mammalian lung. These factors include both proteins and non-proteinaceous molecules. This chapter will focus on two topics. First will be the type II protein secretion system that elaborates a large number and wide variety of enzymes, some of which have recently been implicated as factors that promote infection. Second will be the export of siderophores, non-protein, high-affinity iron chelators that stimulate growth in low-iron conditions.

Introduction

L. pneumophila secretes many different factors that promote environmental survival and growth with the mammalian host. These factors include both proteins and non-proteinaceous molecules. Among the exoproteins, there are a variety of degradative enzymes, including ones with pathogenic function. In L. pneumophila, as in other Gram-negative bacteria, the secretion of exoproteins is a complex process that requires transport across the inner membrane, the periplasmic space, and finally the outer membrane. Gram-negative organisms can have as many as six secretion systems that permit the export of proteins from within the bacterial cell to the extracellular milieu and/or into target host cells; i.e., type I, II, III, IV, V, or VI secretion (Kostakioti et al., 2005; Economou et al., 2006). At least two of these systems are in operation in L. pneumophila (Bruggemann et al., 2006a). A great deal of work has shown that both type II and type IV systems are functional and critical for L. pneumophila. The type II system will be the primary focus of this chapter, whereas the type IV systems will be covered in Chapter 9. Recent genomic sequencing suggests that type I and type V secretion systems may also exist in strains of L. pneumophila (Jacobi and Heuner, 2003; Cazalet et al., 2004). However, whether these systems are functional remains to be determined (Lammertyn and Anne, 2004; Bruggemann et al., 2006a). That L. pneumophila has a flagellar system that is related to type III secretion was discussed in Chapter 6. Turning to the export of non-protein factors, a novel siderophore is implicated in L. pneumophila iron acquisition. This iron chelator, named legiobactin, will be discussed later in this chapter. read more ...

Type II protein secretion

The mechanism of Gram-negative type II secretion

Type II protein secretion (T2S) is a two-step process (Fig. 8.1) (Johnson et al., 2006). In the first step, proteins destined for secretion are translocated across the inner membrane by the Sec pathway or, in fewer instances, the Tat pathway, and then N-terminally cleaved by signal peptidase. In the second step, the proteins are transported from the periplasm to the cell's exterior by a complex of proteins specifically dedicated to T2S, ultimately involving passage of the proteins through an outer membrane pore. The mechanism by which proteins are recognized for entry into the T2S apparatus is not understood but likely involves tertiary structural determinants.

Evolutionarily speaking, the T2S apparatus is related to the type 4 pilus apparatus (Peabody et al., 2003). The T2S apparatus consists of 12 core proteins; i.e., a cytoplasmic ATPase (T2S E), three inner membrane proteins that create a platform and binding site for the ATPase (T2S F, L, M), major and minor 'pseudopilins' which form a pilus-like structure that can span the periplasm (T2S G, H, I, J, K), an inner membrane peptidase that cleaves pre-pseudopilins and then methylates the pseudopilins prior to their integration into the apparatus (T2S O, which is also commonly known as PilD), an outer membrane 'secretin' that oligomerizes to form the exit pore (T2S D), and a protein that may be involved in substrate recognition and/or secretin interactions (T2S C) (Peabody et al., 2003; Filloux, 2004; Johnson et al., 2006). In summary, proteins that are destined for T2S are recognized in the periplasm and then delivered to the T2S apparatus, whereupon, using energy generated at the inner membrane, a pilus-like structure behaves like a piston to push the proteins out through an outer membrane pore (Fig. 8.1). Analysis of sequenced genomes reveals that T2S genes are common but not universal within Gram-negatives (Cianciotto, 2005). Functional studies indicate that T2S promotes the physiology of various environmental bacteria as well as the virulence of numerous human, animal, and plant pathogens (Cianciotto, 2005). In many ways, though, work in L. pneumophila has given us the broadest appreciation of the biological significance of T2S. read more ...

The discovery of type II secretion genes in L. pneumophila

The first indication that L. pneumophila employs T2S was the discovery of a pilBCD locus, encoding the pseudopilin peptidase PilD (T2S O), along with PilB and PilC, which are proteins that are generally only associated with type 4 pilus biogenesis (Liles et al., 1998). Next, it was shown that mutation of pilD in serogroup-1 strain 130b abolished secretion, as evidenced by the loss of proteins in mutant culture supernatants visualized by 1D-SDS-PAGE (Liles et al., 1999). Although pilD transcripts are produced at 37°C, Northern blot and lacZ gene fusion analyses demonstrated that the gene is more highly expressed at lower temperatures such as 30°C (Liles et al., 1998; Söderberg et al., 2004). A study of strain Philadelphia-1 then revealed the presence of lspFGHIJK, predicted to encode the T2S FGHIJK proteins (Hales and Shuman, 1999). As was seen with mutation of pilD, inactivation of lspGH resulted in loss of protein secretion. In two further studies of strain 130b, genes encoding homologues of T2S DE (lspDE), C (lspC), and LM (lspLM) were reported, with mutational analysis of lspDE confirming their role in secretion activity (Rossier and Cianciotto, 2001; Rossier et al., 2004). Taken together, these studies indicated that L. pneumophila has a complete and functional T2S system. This was confirmed when the complete genomes of serogroup-1 strains Philadelphia 1, Paris, and Lens were published (Cazalet et al., 2004; Chien et al., 2004; Cianciotto, 2005). Southern hybridizations have shown that T2S genes are also in a variety of other serogroups of L. pneumophila as well as other species of Legionella (Rossier et al., 2004). A variety of T2S-specific mutants of L. pneumophila have been constructed and characterized; in all cases, the mutants replicate normally in standard buffered yeast extract (BYE) broth at 37°C (Rossier and Cianciotto, 2001; Rossier et al., 2004). T2S-specific mutants also have a typical efficiency of plating on buffered charcoal yeast extract (BCYE) agar at 37°C, but they do show slightly altered colony morphology (Rossier and Cianciotto, 2001; Rossier et al., 2004).

Sec translocation in L. pneumophila

The presence and functioning of T2S-specific genes in L. pneumophila strongly implied that there must also be a mechanism for translocating Legionella proteins across the inner membrane prior to their entry into and secretion through the T2S apparatus. There was little doubt, though, that Sec provides this function, because of its seemingly universal conservation within bacteria (van der Sluis and Driessen, 2006). Indeed, subsequent genome sequencing confirmed that L. pneumophila encodes a complete Sec system for translocation across the inner membrane (Cazalet et al., 2004; Chien et al., 2004; Lammertyn and Anne, 2004).

Like other Gram-negative proteobacteria, L. pneumophila contains the machinery for both co-translational and post-translational translocation. In co-translational translocation, nascent polypeptides are delivered by the signal recognition particle, with the aid of YidC, to an inner membrane channel composed of the trimeric SecYEG complex (Fig. 8.1) (Lee and Schneewind, 2001; Robson and Collinson, 2006). In post-translational translocation, the cytoplasmic SecA ATPase and SecB chaperone deliver newly made protein to SecYEG in a process that also requires SecDF and YajC (Lee and Schneewind, 2001; Robson and Collinson, 2006). SecM, a regulator of SecA expression in E. coli and other Enterobacteriales (van der Sluis and Driessen, 2006), is absent from L. pneumophila. As expected, L. pneumophila encodes the inner membrane type I signal peptidase (LepB) that cleaves the N-terminus from proteins as they emerge, via Sec, on the periplasmic face of the inner membrane (Lammertyn et al., 2004). The type II signal peptidase that cleaves translocated lipoproteins has also been characterized in L. pneumophila (Geukens et al., 2006). Given the overwhelming evidence obtained from the study of other bacteria, there is no doubt that the Sec pathway is essential for L. pneumophila growth, providing the main conduit for many proteins to localize to the periplasm, outer membrane, and/or extracellular milieu. Indeed, recent in silico analysis, using the SignalP algorithm, has determined that there are as many as 576 proteins that contain a typical signal peptide and are potential substrates for Sec translocation (DebRoy et al., 2006b). Additional analyses using PSORTb and PA-SUB, which are designed to further predict cellular localization, suggested that there might be 60 and perhaps even more proteins that are fully secreted into the extracellular space, with presumably a significant portion of those being handled by the T2S system (DebRoy et al., 2006b).

Twin arginine translocation in L. pneumophila

Genome sequencing also confirmed that L. pneumophila possesses the genes encoding twin-arginine translocation (Tat), another inner membrane translocation pathway that is conserved in many organisms (Cazalet et al., 2004; Chien et al., 2004; De Buck et al., 2004; Rossier and Cianciotto, 2005). Although, for years, Tat had been viewed as a system for localizing periplasmic enzymes, the study of phospholipase C enzymes in Pseudomonas aeruginosa revealed that Tat substrates can also feed into the T2S system for extracellular secretion (Ochsner et al., 2002). Thus, the presence of Tat in L. pneumophila has potential significance for Legionella T2S.

Unlike the Sec system, the Tat machinery promotes the transport of fully folded proteins across membranes (Fig. 8.1) (Lee et al., 2006; Sargent et al., 2006). Tat is mediated by three integral membrane proteins, named TatA, TatB, and TatC. Although the signal peptides of Tat-dependently secreted proteins resemble Sec-dependent signal peptides in their overall structure, they differ in several ways from their Sec counterparts. First, they have a twin arginine consensus motif (RRXΦΦ, where Φ is a hydrophobic residue), with that motif being followed by a region that is less hydrophobic than that of Sec signal peptides. Second, basic residues, which could serve as a Sec-avoidance signal, are commonly found just before the signal peptidase cleavage site. Finally, Tat signal peptides can be much longer than the Sec signal sequences. In the current E. coli model, Tat is viewed as a three-step event (Fig. 8.1) (Sargent et al., 2006). First, the twin-arginine signal peptides are bound by an inner membrane complex consisting of TatB and TatC, with TatC probably being the main factor in peptide recognition. Then, a complex consisting of TatA is recruited to the site in a process that is dependent upon the membrane's electrochemical gradient. Finally, the folded exoprotein passes through a channel in the TatA complex.

Once the tatA, tatB, and tatC genes were found in L. pneumophila, RT-PCR analysis revealed that they are expressed during both extra- and intracellular growth (De Buck et al., 2004). Since L. pneumophila tatB mutants grow normally in standard BYE, Tat is not required for general extracellular growth (Rossier and Cianciotto, 2005). However, L. pneumophila tat mutants are defective for growth in iron-limiting conditions and during intracellular infection of macrophages and amoebae, indicating that Tat substrates are critical for Legionella survival under specialized conditions, including those that exist during infection (De Buck et al., 2005; Rossier and Cianciotto, 2005). As regards more specific effects of Tat, it was demonstrated that L. pneumophila Tat is required for cytochrome c-dependent respiration and the associated processing of the cytochrome c reductase PetA (Rossier and Cianciotto, 2005). In silico analysis that looked for the presence of twin arginine residues in signal peptides revealed ca. 35 more putative Tat substrates, which are predicted to be involved in a variety of other cellular processes (De Buck et al., 2004; Rossier and Cianciotto, 2005). read more ...

Factors secreted by the type II system of L. pneumophila

With the knowledge that L. pneumophila possesses the full set of T2S-specific genes, as well as the Sec and Tat machinery for delivering proteins into the periplasm in order that they might then enter the secretion apparatus, we can now focus on the factors that are secreted by Legionella T2S. By 2005, at least eleven enzymes were determined to be secreted via the L. pneumophila T2S system (Rossier et al., 2004; Banerji et al., 2005; Cianciotto, 2005). This conclusion was based upon the loss of enzymatic activities from the culture supernatants of lspDE, lspF, lspG, lspGH, or pilD mutants grown in standard BYE broth at 37°C (Hales and Shuman, 1999; Liles et al., 1999; Aragon et al., 2000; Rossier and Cianciotto, 2001; Rossier et al., 2004). The T2S-dependent activities include tartrate-sensitive and tartrate-resistant acid phosphatases, phospholipase A, phospholipase(s) C, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase (GCAT), mono-, di-, and triacylglycerol lipases, ribonuclease, and protease (Hales and Shuman, 1999; Liles et al., 1999; Aragon et al., 2000; Aragon et al., 2001; Flieger et al., 2001b; Rossier and Cianciotto, 2001; Aragon et al., 2002; Flieger et al., 2002; Rossier et al., 2004; Banerji et al., 2005). Because mutants specifically lacking the type 4 pilus are not defective for these activities, the altered secretion of the pilD mutants is due to the loss of T2S (Rossier and Cianciotto, 2001; Rossier et al., 2004). Several of the secreted enzymatic activities have now been identified in other species of Legionella (Flieger et al., 2001a; Rossier et al., 2004).

In some cases, the structural genes (proteins) encoding T2S-dependent secreted activities have been identified. These include map (Map) for the tartrate-sensitive acid phosphatase (Aragon et al., 2001), plcA (PlcA) for phospholipase C activity (Aragon et al., 2002), plaA (PlaA) for the lysophospholipase A (Flieger et al., 2002), plaC (PlaC) for GCAT (Banerji et al., 2005), lipA (LipA) and lipB (LipB) for mono- and triacylglycerol lipases (Aragon et al., 2002), and proA/msp (ProA/Msp) for the zinc metalloprotease (Hales and Shuman, 1999; Liles et al., 1999). Notably, mutations in any one of these genes do not completely abolish the corresponding activity. Accordingly, L. pneumophila appears to have more than one secreted phosphatase, phospholipase C, lysophospholipase A, lipase, and protease/peptidase, and T2S likely mediates the secretion of more than eleven proteins. The analysis of supernatants from proA mutants demonstrates that some secreted proteins are subject to cleavage and perhaps activation by the T2S-dependent metalloprotease (Flieger et al., 2002; Banerji et al., 2005). By virtue of a twin-arginine in its signal peptide, PlcA of L. pneumophila strain 130b was predicted to be a Tat substrate; indeed, mutations in tatB diminish secreted phospholipase C activity in that strain (Rossier and Cianciotto, 2005).

To further define the proteins secreted by L. pneumophila T2S, proteins in wild type and lsp mutant supernatants were compared by two-dimensional PE, and then mass spectrometry was used to identify the secreted proteins (DebRoy et al., 2006b). Twenty-seven proteins present for wild type but absent or greatly reduced for an lspF mutant were identified. Three of these (i.e., ProA, PlaA, and Map) were previously defined as T2S exoproteins, as noted above. A fourth protein was identified as a ribonuclease, most likely representing the T2S-dependent ribonuclease activity that had been reported before, but for which the gene had been unknown. Of the remaining 23 proteins, 13 were also predicted to contain a signal sequence and are quite likely to represent new type II dependent exoproteins. Two of these were annotated in the database as a leucine aminopeptidase and a chitinase, assignments subsequently confirmed by mutational analysis of the cognate genes (DebRoy et al., 2006b; Rossier and Cianciotto, unpublished). Four others were annotated as hypothetical proteins but having similarity to bacterial enzymes; they included an amidase, another aminopeptidase, a cysteine protease, and an endoglucanase/cellulase. Two others were most similar to eukaryotic proteins, i.e. one had collagen-like repeats, and the other homology to a eukaryotic zinc proteinase. The remaining five proteins that were clearly T2S dependent were annotated as hypothetical with no similarities to any known protein or domain in the database and thus may represent novel activities. Three more proteins, IcmX, LvrE, and a VirK-like protein, identified in wild-type but not mutant supernatants (Matthews and Roy, 2000; De Buck et al., 2005; Albert et al., 2006; DebRoy et al., 2006b), had signal sequences, but their connection to T2S seems more complicated. Indeed, IcmX and LvrE are known for their links, respectively, to the Dot/Icm and Lvh type IV systems of L. pneumophila (Brand et al., 1994; Segal et al., 1999; Matthews and Roy, 2000; Ridenour et al., 2003), and VirK is associated with a type IV secretion system in Agrobacterium tumefaciens (Kalogeraki and Winans, 1998; Li et al., 2005). These data suggest a possible connection between type II and IV secretion. Alternately, although the genes encoding IcmX, LvrE, and the VirK-like protein are linked to type IV secretion genes, the secretion of the proteins may in fact occur by T2S.

The results of the recent proteomic analysis combined with the earlier assessments of culture supernatant enzymatic activities indicate that the number of proteins secreted by the L. pneumophila T2S system is at least 25. However, the total output of the L. pneumophila T2S system is likely greater, for several reasons: i) not every protein observed by 2D-PE was submitted for identification by mass spectroscopy, ii) some T2S effectors might have been missed because of low-level expression or degradation due to the processing of the sample or the action of a secreted protease, iii) the supernatant samples that were examined were from bacteria cultured under only a single growth condition, iv) an in silico screen of the L. pneumophila genome revealed 60 proteins that are predicted to be extracellular by at least one programme; and 13 meet the criteria of two programmes (DebRoy et al., 2006b). Thus, the most likely scenario is that the L. pneumophila T2S processes between 25 and 60 substrates. Even at 25, the experimentally defined catalogue of L. pneumophila T2S effectors is the largest known in bacteria (Cianciotto, 2005; DebRoy et al., 2006b).

Clearly, L. pneumophila secretes a variety of factors via its T2S system. Several of the types of enzymes observed, including proteolytic enzymes, lipolytic enzymes, chitinases, and phosphatases, and potential cellulases, are also secreted by other bacterial T2S systems (Cianciotto, 2005). In some cases, the T2S-dependent effectors of L. pneumophila are actually closely related to the secreted proteins of other bacteria; e.g. ProA/Msp is homologous with well characterized proteases from a variety of organisms, and PlcA is highly related to a new type of phospholipase C found in Pseudomonas (Aragon et al., 2002). But, the type II secretome of L. pneumophila also includes proteins that show their greatest similarity to eukaryotic proteins; e.g. the Map acid phosphatase, a collagen-like protein, and a eukaryotic-like proteinase (Aragon et al., 2001; DebRoy et al., 2006b). L. pneumophila is also unique in its secretion of an RNAse and perhaps an amidase. Perhaps most significantly, a number of the Legionella exoproteins do not bear any similarity to known proteins, raising the possibility of there being novel effectors secreted by T2S. From proteomic data and in silico predictions, the type of proteins that appear to be most represented in the T2S repertoire are proteases/peptidases, a finding consistent with the fact that amino acids are the main source of carbon and energy for broth-grown L. pneumophila (George et al., 1980). The legionellae can also catabolize carbohydrate-derivatives (Weiss et al., 1980; Tesh et al., 1983; Bruggemann et al., 2006b), and the putative Lsp exoproteins do also include proteins with predicted glycosidase activity such as a eukaryotic-like glucoamylase. Based on both the number of proteins uncovered and the types of factors detected, the analysis of L. pneumophila highlights more than ever the impact that T2S has on bacterial function.

A connection between Mip and type II secretion

The recent study of L. pneumophila has given new insight into the mechanisms of secretion, i.e. the involvement of Mip, a surface associated peptidylproline cis-trans isomerase (PPIase), in the elaboration of secreted activity (DebRoy et al., 2006a). Among the activities that are diminished in lsp mutant supernatants is the ability to hydrolyse p-nitrophenol phosphorylcholine (p-NPPC), and in many bacteria, p-NPPC hydrolysis is attributed to phospholipase C enzymes (Merino et al., 1999; Terada et al., 1999). However, since mutation of plcA only partly diminished secreted p-NPPC hydrolase activity (see above), it had been hypothesized that L. pneumophila secretes another phospholipase C (Aragon et al., 2002; Rossier and Cianciotto, 2005). When a screen was done to identify other factors that promote secreted hydrolase activity, a mip mutant was isolated (DebRoy et al.,
2006a).

Although Mip, originally identified for its role in promoting intracellular infection and virulence, is one of the most heavily studied proteins in Legionella (Cianciotto et al., 1989; Cianciotto et al., 1990; Cianciotto and Fields, 1992; Fischer et al., 1992; Ludwig et al., 1994; Cianciotto et al., 1995; Wintermeyer et al., 1995; Helbig et al., 2001; Riboldi-Tunnicliffe et al., 2001; Kohler et al., 2003), its molecular target(s) had remained obscure. Five independently derived mip mutants showed a 40-70% reduction in secreted activity, and the defects were complemented by providing mip in trans. Thus, Mip promotes the presence of a p-NPPC hydrolase activity in culture supernatants. When supernatants were examined by chromatography, the p-NPPC hydrolase activity associated with Mip proved to be T2S-dependent but distinct from PlcA, indicating that Mip promotes the elaboration of a new T2S-exoprotein.

Two hypotheses are proposed to account for these observations (DebRoy et al., 2006a). In the first scenario, Mip is involved in the extracellular release of an active enzyme that has p-NPPC hydrolase activity; Mip could act directly on the exoprotein, or it might promote the maturation of proteins that form the secretion pathway. In the second scenario, Mip interacts with a newly secreted protein and causes changes that convert the protein from being enzymatically inactive to active. These data are the first evidence for a target for Mip and the first case of a surface PPIase being linked to the secretion or activation of proteins beyond the outer membrane (DebRoy et al., 2006a).

The role of type II secretion in L. pneumophila pathogenesis

L. pneumophila T2S mutants (i.e., derivatives containing an insertion mutation in lspDE, lspF, lspG, lspK, or pilD) show a reduced ability to infect human macrophages, including the U937 cell line and peripheral blood mononuclear cell-derived macrophages (Liles et al., 1999; Polesky et al., 2001; Rossier and Cianciotto, 2001; Rossier et al., 2004). In the U937 cells, the mutants displayed a 10-fold reduced yield at 48 hours post inoculation (Polesky et al., 2001; Rossier and Cianciotto, 2001; Rossier et al., 2004). In contrast, an lsp mutant did not grow in macrophages derived from peripheral blood cells, suggesting that the magnitude of the mutant phenotype is influenced by the nature of the host cell. Indeed, when tested in HL60 cells, an lspGH mutant behaved like wild type (Hales and Shuman, 1999). The reduced infectivity of mutants was complemented by reintroduction of the corresponding pilD or lsp genes (Rossier et al., 2004). Since mutants specifically lacking type 4 pili are not defective for macrophage infection, the infectivity defect of the pilD mutant is due to the loss of T2S (Rossier et al., 2004). Beyond their apparent defect in intracellular replication, Lsp mutants, under some growth conditions, exhibit a defect in invasion (Polesky et al., 2001). The T2S mutants are not impaired for the induction of apoptosis or a pore-forming activity linked to egress from host cells (Molmeret et al., 2002; Zink et al., 2002). Together, these data indicate that T2S promotes L. pneumophila infection of macrophages.

Recently, it has been shown that lsp mutants are defective in an animal model of Legionnaires' disease (Rossier et al., 2004). Upon intratracheal inoculation into A/J mice, lspDE, lspF and pilD mutants, but not pilus mutants, display a reduced ability to grow in the lung, as measured by competition assays. The lspF was also defective in an in vivo growth kinetics assay; i.e., whereas wild type increased at least 10-fold in the lungs, the T2S mutant gave no evidence of replication. The examination of the mouse sera revealed that type II-secreted proteins are expressed in vivo. Therefore, L. pneumophila T2S is an important virulence factor. The reduced survival of the lsp mutants in the lung is likely due, at least in part, to reduced growth in alveolar macrophages. However, that mutant numbers do not increase in the lungs, whereas in vitro they do, although not optimally suggests that the lsp mutants are also defective for extracellular processes that are operative in the lungs, such as combating other aspects of the host response. Together, these studies are the first documentation of a role for T2S in intracellular infection and in an animal model of disease.

Mutants that lack particular T2S effectors have also been tested for alterations in infection. The map, plcA, plaA, plaC, lipA, lipB, proA/msp and chiA mutants all grow normally in macrophages in vitro, indicating that the Map tartrate-sensitive acid phosphatase, PlcA phospholipase C, PlaA lysophospholipase A, PlaC GCAT, LipA and LipB lipases, ProA zinc metalloprotease, and ChiA chitinase are not required for macrophage infection (Aragon et al., 2001; Aragon et al., 2002; Flieger et al., 2002; Banerji et al., 2005; DebRoy et al., 2006b). These results imply that the T2S pathway secretes a yet-to-be-defined factor that is necessary for macrophage infection. On the other hand, there may be redundancy in the effectors, such that one factor can compensate for the loss of another. Interestingly, the ChiA chitinase mutant proved to be defective when tested in the murine model of Legionnaires' disease (DebRoy et al., 2006b). Following introduction into the lung, the mutant had a ca. 4-fold deficiency compared to wild type. That independently derived mutants behaved similarly indicated that the defects were due to loss of monocistronic chiA and not spontaneous second-site mutation. Western blot analysis using antisera from animals intratracheally inoculated with wild-type bacteria showed that ChiA is expressed in vivo.

Taken together, these data indicate that ChiA is, directly or indirectly, required for optimal survival of L. pneumophila in the lung. ChiA is the first T2S effector to be implicated in Legionella virulence. Other effector mutants tested, including strains lacking acid phosphatase, lipases A and B, lysophospholipase A, metalloprotease, or phospholipase C, displayed no defect in the mice (DebRoy et al., 2006b). Given that the chiA mutant grew normally in macrophages in vitro, ChiA would seem to be critical for more than intracellular infection. That the reduced survival of the mutant in the lung was manifest in the later stages of infection implies that ChiA promotes persistence vs. replication. Since mammals do not have chitin, these observations lead to the hypothesis that there is a chitin-like substance in the lung whose degradation aids bacterial persistence. Alternatively, ChiA might be a bi-functional enzyme that has another, unidentified substrate, whose loss enhances bacterial survival. Clearly, more work is needed to determine whether these or other hypotheses are correct. Yet, already, these results are the first documentation of a protein having chitinase activity also promoting the survival of a pathogen in a mammalian host (DebRoy et al., 2006b). Thus, factors, such as chitinases, that are traditionally viewed as only being relevant in the environment may actually have great importance in infection. read more ...

The role of type II secretion in environmental survival

L. pneumophila lsp and pilD mutants are very defective for intracellular growth in amoebae, including Hartmannella vermiformis and Acanthamoeba castellanii (Hales and Shuman, 1999; Liles et al., 1999; Polesky et al., 2001; Rossier and Cianciotto, 2001; Rossier et al., 2004). Indeed, the mutants show little, if any, evidence of growth in the protozoa, and their reduced infectivity is complementable. Thus, Legionella T2S is required for amoebal infection. As was the case for macrophage infection, studies using mutants lacking particular enzymes have not identified a T2S effector that is key to protozoan infection (DebRoy et al., 2006b). However, given the importance of protozoa for L. pneumophila survival in water, these data establish T2S as a significant promoter of Legionella persistence in the environment. Since infected protozoa may be part of the infective dose (Brieland et al., 1996; Cirillo et al., 1999), the fact that T2S promotes interactions with protozoa also increases its relevance for pathogenesis.

Following upon the finding that L. pneumophila pilD transcripts are more abundant at 30 vs. 37°C (Liles et al., 1998), it was found that pilD mutants have a reduced ability to grow in BYE broth and to form colonies on BCYE agar at lower temperatures (Söderberg et al., 2004). Mutants specifically impaired in T2S are also greatly impaired for colony formation at 25, 17, and 12°C (Söderberg et al., 2004). These growth defects were complemented by reintroduction of the corresponding pilD or lsp gene. In experiments that more closely mimic aquatic habitats, lsp mutants show reduced survival in tap water incubated at 25, 17, 12, and 4°C (M. A. Söderberg and N. P. Cianciotto, unpublished). The mutants display improved growth at 25°C when plated next to a streak of wild-type but not mutant bacteria, suggesting that a secreted, diffusible factor promotes low-temperature growth (Söderberg et al., 2004). Indeed, growth stimulation could be seen using supernatants from wild type. Mutants lacking the known acid phosphatase, lipases, phospholipase C, lysophospholipase A, or protease grow normally at 25°C, suggesting the existence of a critical, yet-to-be-defined exoproteins(s) (Söderberg et al., 2004). In sum, these data show that L. pneumophila replicates at temperatures below 20°C and that a T2S system can stimulate growth at low temperatures.

Concluding comments

Studies with macrophages, animals, protozoa, and low-temperature conditions illustrate how T2S promotes L. pneumophila growth in a mammalian host and in environmental niches. Much more work is needed to ascertain which T2S effectors promote growth in the disparate niches and how they function. Nonetheless, L. pneumophila already provides the best-characterized T2S system in terms of showing its overall significance for bacterial physiology, pathogenesis, and ecology (Cianciotto, 2005; DebRoy et al., 2006b).

The export of siderophores

L. pneumophila and iron

Iron is a key factor in L. pneumophila growth (Reeves et al., 1981). This iron requirement was first thought to be 3-13 µM for minimal growth and >20 µM for optimal growth (Reeves et al., 1983; Johnson et al., 1991; Mengaud and Horwitz, 1993). But, recent work shows it to be <1 µM (James et al., 1995; Liles et al., 2000). Upon incubation with ⁵⁵FeCl₃, L. pneumophila takes up iron in an energy-dependent, protease-resistant process (Johnson et al., 1991). As in other bacteria, iron serves as a cofactor in a variety of enzymes (Mengaud and Horwitz, 1993). The ability of L. pneumophila to replicate in the mammalian host is also highly dependent on iron. For example, human macrophages treated with iron chelators do not support bacterial growth, and interferon-γ inhibits intracellular growth by limiting iron (Byrd and Horwitz, 2000; Viswanathan et al., 2000). Additionally, some murine macrophages become permissive for Legionella infection only after the addition of iron (Gebran et al., 1994). Furthermore, iron supplementation increases the susceptibility of animals to infection, and legionellae grown under iron-limiting conditions exhibit reduced virulence (James et al., 1995).

The first genetic data on the role of iron in Legionella was the identification of the gene for the transcriptional repressor Fur (Hickey and Cianciotto, 1994). Its repressive activity is, not surprisingly, highest in legionellae grown in iron-rich media (Hickey and Cianciotto, 1994, 1997). Since L. pneumophila fur could not be insertionally inactivated, Fur is likely essential for Legionella aerobic growth. The importance of L. pneumophila fur is evident from the identification of multiple iron- and Fur-regulated genes (Hickey and Cianciotto, 1997). Although the importance of iron for Legionella has always been clear, the mechanisms used by the bacterium to acquire and transport iron have been rather elusive. Traditional biochemical methods nicely determined that L. pneumophila encodes a cytoplasmic and a periplasmic ferric reductase (James et al., 1997; Johnson et al., 1991; Poch and Johnson, 1993). Nevertheless, as will be described momentarily, proof of the existence of a secreted siderophore was nearly 25 years in waiting.

The discovery of a Legionella siderophore

In 1983, it was reported that L. pneumophila does not make siderophores (Reeves et al., 1983). This conclusion was based largely on results from assays that detect typical catecholate and hydroxamate structures. Eight years later, the question of Legionella siderophores was revisited using the CAS assay, which detects iron chelators regardless of their structure (Goldoni et al., 1991). CAS reactivity was detected in statically grown cultures, suggesting the existence of a non-catecholate, -hydroxamate siderophore. Yet, a later study determined that the CAS reactivity was due to cysteine in the medium (Liles and Cianciotto, 1996). When supernatants where obtained from cultures made with cysteine-free media, no CAS reactivity was seen (Liles and Cianciotto, 1996; James et al., 1997). In 2000, however, it was shown that L. pneumophila could excrete a high-affinity iron-chelator (legiobactin) when grown at 37°C in a low-iron, chemically defined medium (Liles et al., 2000). Importantly, the CAS reactivity was only observed when cultures are inoculated with bacteria that had been grown to log or early stationary phase. Inocula derived from late-stationary phase cultures, despite growing in the CDM, do not display the siderophore-like activity. CAS reactivity was observed for multiple serogroup 1 strains as well as isolates representing all nine of the other serogroups tested (Liles et al., 2000). The CAS reactivity has been studied further in L. pneumophila serogroup-1 strain 130b, and all data signal that it is a true siderophore (Liles et al., 2000). For example, CAS reactivity correlates with enhanced aerobic growth in an iron-deplete defined medium, and the chelating activity is subject to iron-repression. Moreover, the CAS reactivity is less than 1 kDa in size, is resistant to heat and proteases, and appears rapidly and intensely. Finally, CAS-positive supernatants stimulate the growth of wild-type in BCYE agar containing otherwise inhibitory concentrations of iron chelator (Allard et al 2006). Most other Legionella species tested also secrete CAS reactivity, although it cannot be concluded yet that they are producing legiobactin (Liles et al., 2000; Starkenburg et al., 2004). read more ...

Genes involved in siderophore production

Two linked genes, lbtA and lbtB, have recently been identified as being involved in the expression of legiobactin (Allard et al., 2006). The lbtA gene is predicted to encode a protein with homology to siderophore synthetases, including enzymes involved in the biosynthesis of aerobactin in E. coli, alcaligin in Bordetella species, and rhizobactin 1021 in Sinorhizobium meliloti (Allard et al., 2006). Very recent studies have also uncovered an LbtA-like gene in Francisella tularensis (Deng et al., 2006; Sullivan et al., 2006). LbtA is also related to FrgA, a L. pneumophila protein that had previously been shown to be iron-regulated but not required for siderophore expression (Hickey and Cianciotto, 1997; Liles et al., 2000; Allard et al., 2006). The lbtB gene encodes a protein that is homologous with members of the major facilitator superfamily of multidrug efflux pumps. Significantly, members of this superfamily have recently been implicated in siderophore export, including proteins required for the export of enterobactin in E. coli, protochelin in Azotobacter vinelandii, achromabactin in E. chrysanthemi, and alcaligin in Bordetella species (Furrer et al., 2002; Brickman and Armstrong, 2005; Franza et al., 2005). Thus, LbtA is predicted to be involved in the biosynthesis of legiobactin, and LbtB in its export. Mutants inactivated for lbtA or lbtB are impaired in legiobactin expression, producing 40-70% less CAS reactivity in iron-deplete CDM (Allard et al., 2006). In bioassays, mutant supernatants, unlike those of wild type, do not support the growth of iron-limited wild-type bacteria or a ferrous iron transport mutant. The lbtA mutant was also defective for growth in media containing the iron chelator citrate, indicating that legiobactin is needed in conditions of severe iron limitation. The iron (III) K_s for legiobactin has been estimated to be between 10¹¹ and 10³¹ (Allard et al., 2006). Trans-complementation of the lbt mutants restored both legiobactin expression as measured by the CAS assay and bioassays and bacterial growth in citrate-containing media (Allard et al., 2006). Fur boxes precede lbtAB, suggesting that Fur mediates the iron-regulation of legiobactin. lbtA is widely distributed among Legionella strains (Allard et al., 2006).

Bacteria usually have large siderophore operons encoding a number of biosynthetic enzymes and a ferrisiderophore receptor; e.g. there appear to be six biosynthetic genes for rhizobactin, and three to five for alcaligin (Challis, 2005). The L. pneumophila system seems different. There is a gene (lbtC) downstream of lbtAB that is predicted to be another MFS member, but mutations in it do not alter siderophore expression (Allard et al., 2006). Furthermore, examination of the genome reveals no other obvious candidate legiobactin genes unlinked to lbtABC. Hence, the biosynthesis of legiobactin may be simple, potentially involving LbtA and one or two precursors. On the other hand, more legiobactin genes may exist, but they would appear to be unique in content and location. In a similar vein, the uptake of legiobactin would appear to be mediated by a novel process since the L. pneumophila genome does not contain TonB, the membrane spanning protein that has been typically implicated in siderophore import (Koebnik, 2005).

The role of siderophores in infection and ecology

To begin to assess the role of siderophores in Legionella infection and environmental persistence, lbtAB mutants were tested for their ability to infect macrophages and Hartmannella amoebae (Allard et al., 2006). Although the lbtA gene was shown to be expressed intracellularly, the mutants were not defective for macrophage or amoeba infection. The mutant was also not impaired for infection of the mouse lung, suggesting that legiobactin is not required for infection (Allard et al., 2006). These data do not, however, indicate that siderophores are not relevant for L. pneumophila: a second siderophore could compensate for the loss of legiobactin, as is very often the case in other bacteria. Interestingly, FrgA mutants are defective in macrophages (Hickey and Cianciotto, 1997), supporting the notion that L. pneumophila has a second siderophore that promotes intracellular infection. The fact that mutations in lbtAB do not completely abolish CAS reactivity further suggests that additional iron chelators are secreted by L. pneumophila.

Concluding comments

With the discovery of legiobactin, we now have, after years of disbelief, evidence of a Legionella siderophore. It is likely that the novel influence of the bacterial inoculum on siderophore expression is the primary reason that legiobactin was not found earlier. It is reasonable that L. pneumophila produces a siderophore(s), since many other aquatic bacteria produce these iron scavengers. Given the phenotype of lbtAB mutants, legiobactin is likely to be most critical for extracellular, rather than intracellular, growth; e.g. it might be vital for growth in aquatic environments such as biofilms. Clearly, further work is needed in this area as well as to address the question of whether L. pneumophila and the other legionellae export siderophores besides legiobactin. read more ...

Acknowledgements

The author wishes to thank both past and present lab members for all of their work and dedication. Our studies are supported by NIH grants AI34937 and AI43987.

from Legionella: Molecular Microbiology
See also: Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
See also: Bacterial Polysaccharides: Current Innovations and Future Trends

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