Life Cycle of L pneumophila

When planktonic, transmissive L. pneumophila are engulfed by phagocytic cells, the bacteria avoid lysosomal degradation and instead establish vacuoles isolated from the endocytic network (Fig. 6.1). To gauge nutrient conditions within its host cell, L. pneumophila employs the Pht family of transporters (Sauer et al. 2005). If vacuolar conditions are favorable, the post-transcriptional regulator CsrA, and perhaps the sRNA chaperone Hfq, suppress transmissive traits and promote intra-cellular replication (Fig. 6.2) (Fettes et al. 2001; Molofsky and Swanson 2003; McNealy et al. 2005). When nutrients are depleted, bacterial replication halts and the ribosomal enzyme RelA produces (p)ppGpp (Hammer and Swanson 1999; Zusman et al. 2002). The accumulation of (p)ppGpp in the bacterial cytosol either

Life Cycle Legionella Pneumophila

Figure 6.1. Life cycle of L. pneumophila. (1) Transmissive L. pneumophila engulfed by phagocytic cells reside in vacuoles and avoid lysosomal degradation. (2) Under favorable conditions, transmissive bacteria begin to replicate. (3) When nutrients are depleted, replicating bacteria stop dividing and begin to express transmission traits. (4) Microbes may develop into a more resilient and infectious mature intracellular form (MIF). (5) The host cell is lysed and transmissive microbes are released into the environment. (6) L. pneumophila that do not encounter a new host cell probably establish biofilms in water systems and ponds. (7) When microbes encounter a host cell, the cycle begins anew. (8) L. pneumophila cultured in broth to either exponential or stationary phase exhibit many of the traits of the replicative and transmissive forms, respectively. Modified from Molofsky AB and Swanson MS (2004) Mol Microbiol 53(1):29-40.

Figure 6.1. Life cycle of L. pneumophila. (1) Transmissive L. pneumophila engulfed by phagocytic cells reside in vacuoles and avoid lysosomal degradation. (2) Under favorable conditions, transmissive bacteria begin to replicate. (3) When nutrients are depleted, replicating bacteria stop dividing and begin to express transmission traits. (4) Microbes may develop into a more resilient and infectious mature intracellular form (MIF). (5) The host cell is lysed and transmissive microbes are released into the environment. (6) L. pneumophila that do not encounter a new host cell probably establish biofilms in water systems and ponds. (7) When microbes encounter a host cell, the cycle begins anew. (8) L. pneumophila cultured in broth to either exponential or stationary phase exhibit many of the traits of the replicative and transmissive forms, respectively. Modified from Molofsky AB and Swanson MS (2004) Mol Microbiol 53(1):29-40.

directly or indirectly stimulates the LetA/LetS two-component system to relieve CsrA repression of transmissive traits (Hammer and Swanson 1999; Hammer et al. 2002; Molofsky and Swanson 2003). Together, the LetA/LetS system, the enhancer protein LetE, and the alternative sigma factors RpoS, RpoN, and FliA induce traits thought to promote efficient host transmission and survival in the environment, including evasion of phagosome-lysosome fusion, motility, cytotoxicity, sodium sensitivity and resistance to environmental stresses (Bachman and Swanson 2001; Hammer et al. 2002; Lynch et al. 2003; Bachman and Swanson 2004a; Bachman and Swanson 2004b; Jacobi et al. 2004). Under particular conditions, L. pneumophila further develops into the highly resilient and infectious cell type, the mature intracellular form (MIF) (Faulkner and Garduño 2002; Garduño et al. 2002). Eventually, the exhausted host cell lyses, and progeny are released into the environment. While L. pneumophila that fail to find a new phagocyte probably establish complex biofilms, planktonic bacteria that encounter another suitable host can initiate the intracellular life cycle once more.

Biofilm Planktonic

Figure 6.2. A model for regulation of L. pneumophila differentiation. Arrows indicate activation and bars indicate inhibition. Replicative phase regulatory interactions are represented by solid double lines, while transmission phase regulatory pathways are indicated by a single solid line. Speculative interactions are designated by dotted lines.

Figure 6.2. A model for regulation of L. pneumophila differentiation. Arrows indicate activation and bars indicate inhibition. Replicative phase regulatory interactions are represented by solid double lines, while transmission phase regulatory pathways are indicated by a single solid line. Speculative interactions are designated by dotted lines.

3. Broth Model

Broth cultures of L. pneumophila grown to either the exponential or stationary phase exhibit traits similar to replicative and transmissive bacteria, respectively, that are observed in co-cultures with phagocytic cells (Fig. 6.1). While many of the different stages and regulatory elements of the L. pneumophila life cycle were originally discerned by observing synchronous broth cultures, subsequent analysis in eukaryotic cells has supported many of these findings. Likewise, comparison of the transcription profiles of L. pneumophila cultured in broth and in Acanthamoeba castellanii has revealed that 84% of replicative phase genes and 77% of transmissive phase genes are upregulated both in vitro and in vivo, thereby confirming the utility of broth culture studies (Brüggemann et al. 2006). Several lines of evidence suggest that the replicative and transmissive phases observed in both broth and eukaryotic cell cultures are reciprocal. For example, when stationary phase L. pneumophila are cultured with eukaryotic cells, they suppress their transmissive traits of cytotoxicity, sodium sensitivity, and motility, and instead replicate profusely (Byrne and Swanson 1998; Alli et al. 2000). Following the replicative period, transmissive traits are induced, and the host cell lyses (Byrne and Swanson 1998; Alli et al. 2000). Similarly, FlaA, Mip, DotH, and DotO proteins, which are known to enhance invasion of eukaryotic cells, are expressed during the entry and exit periods, but not during replication

(Hammer and Swanson 1999; Watarai et al. 2001; Wieland et al. 2002). In contrast, the promoter of CsrA, a repressor of transmission traits, is only active during the replicative period, not during invasion or host cell lysis (Molofsky and Swanson 2003).

While pure bacterial cultures are advantageous for many molecular and biochemical techniques, several characteristics of L. pneumophila have only been observed in vivo, emphasizing the simplicity and limitations of the broth model. For instance, the replication vacuole in A/J mouse macrophages acidifies and L. pneumophila remain acid tolerant, whereas exponentially growing broth cultures are acid sensitive (Sturgill-Koszycki and Swanson 2000). Additionally, bacteria harvested from A. castellanii are more infectious than agar-grown bacteria, suggesting that the intracellular environment of amoebae can affect virulence traits (Cirillo et al. 1999). Moreover, after extended culture in HeLa cells, L. pneumophila differentiates to the cyst-like MIF, a cell type also observed in amoebae and clinical samples, but not in broth culture (Greub and Raoult 2003; Garduño et al. 2002). The substantial differences observed between broth and phagocytic cell cultures highlight the impact of experimental design and lend caution to making inferences based on any single laboratory model.

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