Legionella pneumophila

L. pneumophila is a Gram-negative, non-encapsulated, aerobic bacillus with a single, polar flagellum often characterized as being a coccobacillus. It is aerobic and unable to hydrolyse gelatin or produce urease. It is also nonfermentative. L. pneumophila is neither pigmented nor does it autofluoresce. It is oxidase- and catalase-positive, and produces beta-lactamase. L. pneumophila colony morphology is gray-white with a textured, cut-glass appearance; it also requires cysteine and iron to thrive. It grows on yeast extract in "opal-like" colonies.

L. pneumophila (red chains) multiplying inside Tetrahymena pyriformis

L. pneumophila is a facultative intracellular parasite that can invade and replicate inside amoebae in the environment, especially species of the genera Acanthamoeba and Naegleria, which can thus serve as a reservoir for L. pneumophila. These hosts also provide protection from environmental stresses, such as chlorination.[5]

In the United States, about 2 infections with L. pneumophila appear per 100,000 residents per year[6]. The infections peak in the summer. Within endemic regions, about 4% to 5% of pneumonia cases are caused by L. pneumophila.[7] .

In humans, L. pneumophila invades and replicates inside macrophages. The internalization of the bacteria can be enhanced by the presence of antibody and complement, but is not absolutely required. Internalization of the bacteria appears to occur through phagocytosis. However, L. pneumophila is also capable of infecting nonphagocytic cells through an unknown mechanism. A rare form of phagocytosis known as coiling phagocytosis has been described for L. pneumophila, but this is not dependent on the Dot/Icm secretion system and has been observed for other pathogens.[8] Once internalized, the bacteria surround themselves in a membrane-bound vacuole that does not fuse with lysosomes that would otherwise degrade the bacteria. In this protected compartment, the bacteria multiply.

Legionella-containing vacuole

TEM image of L. pneumophila within a phagocytic cell

For Legionella to survive within macrophages and protozoa, it must create a specialized compartment known as the Legionella-containing vacuole (LCV). Through the action of the Dot/Icm secretion system, the bacteria are able to prevent degradation by the normal endosomal trafficking pathway and instead replicate. Shortly after internalization, the bacteria specifically recruit endoplasmic reticulum-derived vesicles and mitochondria to the LCV while preventing the recruitment of endosomal markers such as Rab5 and Rab7. Formation and maintenance of the vacuoles are crucial for pathogenesis; bacteria lacking the Dot/Icm secretion system are not pathogenic and cannot replicate within cells, while deletion of the Dot/Icm effector SdhA results in destabilization of the vacuolar membrane and no bacterial replication.[14][15]

Legionella is auxotrophic for seven amino acids: cysteine, leucine, methionine, valine, threonine, isoleucine, and arginine [16]. Once inside the host cell, Legionella needs nutrients to grow and reproduce. Inside the vacuole, nutrient availability is low; the high demand of amino acids is not covered by the transport of free amino acids found in the host cytoplasm. To improve the availability of amino acids, the parasite promotes the host mechanisms of proteasomal degradation. This generates an excess of free amino acids in the cytoplasm of L. pneumophila-infected cells that can be used for intravacuolar proliferation of the parasite.

To obtain amino acids, L. pneumophila uses the AnkB bona fide F-Box effector, which is farnesylated by the activity of three host enzymes localized in the membrane of the LCV: farnesyltransferase, Ras-converting enzyme-1 protease, and isoprenyl cysteine carboxyl methyl transferase. Farnesylation allows AnkB to get anchored into the cytoplasmic side of the vacuole.

Once AnkB is anchored into the LCV membrane, it interacts with the SCF1 ubiquitin ligase complex and functions as a platform for the docking of K48-linked polyubiquitinated proteins to the LCV.

The K48-linked polyubiquitination is a marker for proteasomal degradation that releases two- to 24-amino-acid-long peptides, which are quickly degraded to amino acids by various oligopeptidases and aminopeptidases present in the cytoplasm. Amino acids are imported into the LCV through various amino acid transporters such as the SLC1A5 (neutral amino acid transporter). The amino acids are the primary carbon and energy source of L. pneumophila, that have almost 12 classes of ABC transporters, amino-acid permeases, and many proteases, to exploit it. The imported amino acids are used by L. pneumophila to generate energy through the TCA cycle (Krebs cycle) and as sources of carbon and nitrogen.

However, promotion of proteasomal degradation for the obtention of amino acids may not be the only virulence strategy to obtain carbon and energy sources from the host. Type II–secreted degradative enzymes may provide an additional strategy to generate carbon and energy sources.

The determination and publication of the complete genome sequences of three clinical L. pneumophila isolates in 2004 paved the way for the understanding of the molecular biology of L. pneumophila in particular and Legionella in general. In-depth comparative genome analysis using DNA arrays to study the gene content of 180 Legionella strains revealed high genome plasticity and frequent horizontal gene transfer. Further insight in the L. pneumophila lifecycle was gained by investigating the gene expression profile of L. pneumophila in Acanthamoeba castellanii, its natural host. L. pneumophila exhibits a biphasic lifecycle and defines transmissive and replicative traits according to gene expression profiles.[2]

Transformation is a bacterial adaptation involving the transfer of DNA from one bacterium to another through the surrounding liquid medium. Transformation is a bacterial form of sex.[17] In order for a bacterium to bind, take up, and recombine exogenous DNA into its chromosome, it must enter a special physiological state referred to as "competence".

To determine which molecules may induce competence in Legionella pneumophila, 64 toxic molecules were tested.[18] Only six of these molecules, all DNA damaging agents, caused strong induction of competence. These were mitomycin C (which introduces DNA inter-strand crosslinks), norfloxacin, ofloxacin, and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks), bicyclomycin (causes double-strand breaks) and hydroxyurea (causes oxidation of DNA bases). These results suggest that competence for transformation in Legionella pneumophilia evolved as a response to DNA damage.[18] Perhaps induction of competence provides a survival advantage in a natural host, as occurs with other pathogenic bacteria.[17]

Several enzymes in the bacteria have been proposed as tentative drug targets. For example, enzymes in the iron uptake pathway have been suggested as important drug targets.[19] Further, a cN-II class of IMP/GMP specific 5´nucleotidase which has been extensively characterized kinetically. The tetrameric enzyme shows aspects of positive homotropic cooperativity, substrate activation and presents a unique allosteric site that can be targeted to design effective drugs against the enzyme and thus, the organism. Moreover, the enzyme is distinct than its human counterpart making it an attractive target for drug development.[20]

Macrolides (azithromycin or clarithromycin) or fluoroquinolones (levofloxacin or moxifloxacin) are the standard treatment for Legionella pneumonia in humans, with levofloxacin being considered first line with increasing resistance to azithromycin. Two studies support superiority of levofloxacin over macrolides, although not FDA approved.[21]

• Major topic "Legionella pneumophila": free-full text articles in PubMed
• Legionella pneumophila, causative agent of Legionnaire's disease and Pontiac fever at MetaPathogen
• Type strain of Legionella pneumophila at BacDive - the Bacterial Diversity Metadatabase