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 positive- 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.
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. Internalization of the bacteria appears to occur through phagocytosis however L. pneumophila is also capable of infecting non-phagocytic cells through an unknown mechanism. A rare form of phagocytosis known as coiling phagocytosis has been described for L. pneumophila however this is not dependent on the Dot/Icm secretion system and has been observed for other pathogens. 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 Legionella containing vacuole (LCV)
In order for Legionella to survive within macrophages and protozoa it must create a specialized compartment known as the Legionella containing vacuole or LCV. Through the action of the Dot/Icm secretion system, the bacteria is able to prevent degradation by the normal endosomal trafficking pathway and instead replicates. Shortly after internalization, the bacteria specifically recruits 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 vacuole are crucial for pathogensis, bacteria lacking the Dot/Icm secretion system are non-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.
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. 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.
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.
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.
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. 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. 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. Perhaps induction of competence provides a survival advantage in a natural host, as occurs with other pathogenic bacteria.
Macrolides (azithromycin or clarithromycin) or fluoroquinolones (levofloxacin or moxifloxacin) are the standard treatment for Legionella pneumonia in humans with levofloxicin being considered first line with increasing resistance to azithromycin. Two studies support superiority of levofloxacin over macrolides, although not FDA approved. 
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1976 Philadelphia Legionnaires' disease outbreak
The 1976 Legionnaires Disease outbreak also known as Legion Fever, refers to the first known cases of infection by Legionella pneumophila in the United States.
On July 21, 1976, the American Legion opened its annual three-day convention at the Bellevue-Stratford Hotel in Philadelphia, Pennsylvania. More than 2,000 Legionnaires, mostly men, attended the convention. The date and city were chosen to coincide with America’s celebration of the 200th anniversary of the signing of the Declaration of Independence at Philadelphia in 1776.
On July 27, three days after the convention ended, Legionnaire Ray Brennan, a 61-year-old retired Air Force captain and an American Legion bookkeeper, died at his home of an apparent heart attack. Brennan had returned home from the convention on the evening of July 24 complaining of feeling tired. On July 30, another Legionnaire, Frank Aveni, aged 60, also died of an apparent heart attack, as did three other Legionnaires. All of them had been convention attendees. Twenty-four hours later, on August 1, six more Legionnaires died. They ranged in age from 39 to 82, and, like Ray Brennan, Frank Aveni, and the three other Legionnaires, all had complained of tiredness, chest pains, lung congestion, and fever.
Three of the Legionnaires had been patients of Dr. Ernest Campbell, a physician in Bloomsburg, Pennsylvania, who noticed that all three men had been at the Legionnaires convention in Philadelphia. He contacted the Pennsylvania Department of Health. Officials at the American Legion also began getting notices of the sudden deaths of several members, all at the same time. Within a week, more than 130 people, mostly men, had been hospitalized, and 25 had died.
The U.S. Centers for Disease Control and Prevention mounted an unprecedented investigation and, by September, the focus had shifted from outside causes, such as a disease carrier, to the hotel environment itself. In January 1977, the Legionella bacterium was finally identified and isolated and was found to be breeding in the cooling tower of the hotel’s air conditioning system, which then spread it through the building. This finding prompted new regulations worldwide for climate control systems.
Complicating the situation was a fear among the public that the original cluster of 14 cases, six of whom died all within days of each other, represented an outbreak of swine flu. The total number of cases reached 221, and of those, 34 had died. At the time of the outbreak, epidemiological investigation protocols did not include active participation by both the laboratory specialists and investigators. No effective communication existed between scientists in the field interviewing patients, and those in the laboratory testing specimens.
Discovery of Legionella pneumophila
While the Center for Disease Control responded rapidly, as did the Pennsylvania Health Department, it wasn't until nearly a year later that Dr. Joseph McDade made the discovery that a previously identified bacterium was the cause of the outbreak. It had not been considered previously because it was believed to affect only animals. The bacterium was later named Legionella pneumophila.
- 1999 Bovenkarspel legionellosis outbreak
- Legionella pneumophila
- Legionnaires' disease
- List of Legionellosis outbreaks
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