Legionella pneumophila is a thin, aerobic, pleomorphic, flagellated, non-spore forming, Gram-negative bacterium of the genus Legionella. L. pneumophila is the primary human pathogenic bacterium in this group and is the causative agent of legionellosis or Legionnaires' disease.
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 non-fermentative. L. pneumophila is neither pigmented nor does it autofluoresce. It is oxidase- and catalase-positive, and produces beta-lactamase. L. pneumophila has a colony morphology that is gray-white with a textured, cut-glass appearance; it also requires cysteine and iron to thrive. It will grow on yeast extract in "opal-like" colonies.
Cell membrane structure
While L. pneumophila is categorized as a Gram-negative organism, it stains poorly due to its unique lipopolysaccharide-content in the outer leaflet of the outer cell membrane. The bases for the somatic antigen specificity of this organism is located on the side-chains of its cell wall. The chemical composition of these side chains both with respect to components, as well as arrangement of the different sugars, determines the nature of the somatic or O-antigen determinants, which are important means of serologically classifying many Gram-negative bacteria. At least 35 different serovars of L. pneumophila have been described, as well as several other species being subdivided into a number of serovars.
Sera have been used both for slide agglutination studies as well as for direct detection of bacteria in tissues using fluorescent-labelled antibody. Specific antibody in patients can be determined by the indirect fluorescent antibody test. ELISA and microagglutination tests have also been successfully applied.
Legionella stains poorly with gram stain, stains positive with silver, and is cultured on charcoal yeast extract with iron and cysteine.
Obtention of nutrients
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 by not covered by the transport of free amino acids found in the host cytoplasm. In order 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 intra-vacuolar proliferation of the parasite.
To obtain amino acids, Legionella 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 ( Legionella containing vacuole): farnesyltransferase (FTase), Ras-converting enzyme-1 protease (RCE1), and isoprenyl cysteine carboxyl methyl transferase (IcmT). 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 2 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 utilized 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.
L. pneumophila is a facultative intracellular bacterium that can invade and replicate inside amoebae in the environment, which can thus serve as a reservoir for L. pneumophila, as well as provide protection from environmental stresses, such as chlorination.
Frequency of occurrence
In the USA, about 30 infections with L. pneumophila appear per 100,000 residents per year. The infections peak within the summer months. Within endemic regions, about 4% to 5% of pneumonia are caused by L. pneumophila.
In humans, L. pneumophila invades and replicates in macrophages. The internalization of the bacteria can be enhanced by the presence of antibody and complement, but is not absolutely required. A pseudopod coils around the bacterium in this unique form of phagocytosis. 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.
Dot/Icm Type IV Secretion System
The bacteria use a Type IVB secretion system known as Dot/Icm to inject effector proteins into the host. These effectors are involved in increasing the bacteria's ability to survive inside the host cell. L. pneumophila encodes for over 200 "effector" proteins, which are secreted by the Dot/Icm translocation system in order to interfere with host cell processes in order to aid bacterial survival. One key way in which L. pneumophila uses its effector proteins is to interfere with fusion of the Legionella-containing vacuole (LCV) with the host's endosomes, and thus protect against lysis. Knock-out studies of Dot/Icm translocated effectors indicate that they are vital for the intracellular survival of the bacterium, however many individual effector protein are thought to function redundantly, in that single effector knock-outs rarely impede intracellular survival. This high number of translocated effector proteins and their redundancy is likely a result of the bacterium having evolved in many different protozoan hosts.
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 the high genome plasticity and frequent horizontal gene transfer. Further insight in the L. pneumophila life cycle was gained by investigating the gene expression profile of L. pneumophila in Acanthamoeba castellanii, its natural host. L. pneumophila exhibits a biphasic life cycle and defines transmissive and replicative traits according to gene expression profiles.
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