Virulence factors, Pathogenesis and Clinical manifestations of Legionella pneumophila

Legionella pneumophila is a Gram-negative bacterium that can cause a severe pneumonia in humans called Legionnaires` disease (LD) or a mild flu-like illness called Pontiac fever (PF) . The bacterium is widely distributed in natural and artificial aquatic environments, where it parasitizes free-living amoebae . Human infection occurs by inhalation of contaminated aerosols that reach the alveoli and are phagocytosed by alveolar macrophages . However, instead of being killed by these host cells, L. pneumophila is able to survive and replicate within a specialized membrane-bound compartment called the Legionella-containing vacuole (LCV) . This intracellular lifestyle is essential for the pathogenesis of L. pneumophila and requires the expression of a large arsenal of virulence factors that modulate various host cell processes .

Some of the most important virulence factors of L. pneumophila are:

• Heat shock protein 60 (Hsp60): a chaperone protein that enhances the invasion and cytokine expression in macrophages .
• Outer membrane protein (OMP): a porin protein that binds to and delivers packaged materials into the eukaryotic cells and inhibits the fusion of phagosomes with lysosomes .
• Macrophage infectivity potentiator (Mip) protein: a surface-exposed protein that promotes adherence and phagocytosis of L. pneumophila by macrophages .
• Type II secretion system (T2SS): a secretion system that translocates various enzymes and toxins into the extracellular milieu or into the LCV lumen, such as phosphatase, lipase, nuclease, metalloprotease and hemolysin . The T2SS is required for intracellular growth, evasion of host defenses and environmental persistence of L. pneumophila .
• Type IV pili (T4P): hair-like appendages that mediate the entry of L. pneumophila into macrophages and influence the trafficking of the LCV .
• Flagella: whip-like structures that confer motility to L. pneumophila and are involved in bacterial entry, inhibition of host cell apoptosis and modulation of host immune responses .
• Dot/Icm type IV secretion system (T4SS): a secretion system that translocates more than 300 effector proteins into the host cell cytosol or membrane, where they manipulate various cellular pathways to establish and maintain the LCV . The Dot/Icm T4SS is the major virulence determinant of L. pneumophila and is essential for intracellular replication, bacterial entry, inhibition of host cell apoptosis and egress of L. pneumophila from host cells .
• Major outer membrane proteins (MOMPs): surface-exposed proteins that are involved in adherence, intracellular replication, biofilm development and horizontal gene transfer of L. pneumophila .

These virulence factors enable L. pneumophila to exploit its host cells as a niche for replication and dissemination, causing severe lung damage and systemic inflammation in susceptible individuals.

Heat shock protein 60 (Hsp60) is a type of chaperone protein that helps other proteins to fold correctly and prevent aggregation. Hsp60 is also an immunodominant antigen of Legionella pneumophila, the causative agent of Legionnaires` disease and Pontiac fever. Hsp60 is encoded by the htpB gene, which is part of the htpAB operon that also includes htpA, a gene encoding a small heat shock protein.

Hsp60 has been shown to play a role in the invasion and survival of L. pneumophila in macrophages, the primary host cells for this bacterium. Hsp60 binds to the complement component C3b on the bacterial surface, which facilitates the attachment and uptake of L. pneumophila by macrophages through the complement receptor 3 (CR3). Hsp60 also stimulates the production of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6), by infected macrophages. These cytokines may contribute to the clearance of L. pneumophila by activating other immune cells, but they may also cause tissue damage and inflammation in the lungs.

Hsp60 is expressed both in the cytosol and on the membrane of L. pneumophila. The membrane-associated Hsp60 is slightly larger than the cytosolic Hsp60, but they share common epitopes and amino acid sequences. It is possible that L. pneumophila has two hsp60 genes, or that the membrane-associated Hsp60 undergoes post-translational modifications. The localization and function of Hsp60 in different compartments of L. pneumophila are not fully understood.

Hsp60 is a highly conserved protein among bacteria, and it shares homology with GroEL of Escherichia coli and HtpB of Coxiella burnetii. However, Hsp60 also has some unique features that distinguish it from other bacterial Hsp60s. For example, Hsp60 of L. pneumophila has a higher molecular weight (60 kDa) than GroEL of E. coli (57 kDa), and it has a lower affinity for ATP than GroEL. Moreover, Hsp60 of L. pneumophila is more antigenic for human T lymphocytes than GroEL of E. coli, suggesting that it may elicit a stronger immune response.

In summary, Hsp60 is a multifunctional protein that enhances the invasion and cytokine expression of L. pneumophila in macrophages. It may also be involved in other aspects of L. pneumophila pathogenesis, such as stress response, intracellular replication, and antigen presentation.

One of the most important virulence factors of L. pneumophila is the outer membrane protein (OMP), which is a major component of the bacterial cell wall. OMP has several functions that contribute to the pathogenesis of Legionella infections.

First, OMP can bind to various host cell receptors, such as complement receptor 3 (CR3), mannose receptor, and integrins, and facilitate the uptake of the bacteria by phagocytic cells. OMP also mediates the attachment of L. pneumophila to lung epithelial cells and biofilm formation on surfaces.

Second, OMP can deliver packaged materials into the eukaryotic cells through a process called coiling phagocytosis. Coiling phagocytosis is a unique mechanism of bacterial entry that involves the wrapping of pseudopods around the bacteria without forming a complete phagosome. This allows L. pneumophila to inject effector proteins and DNA into the host cytoplasm and modulate various cellular processes, such as gene expression, signal transduction, cytoskeleton dynamics, and vesicle trafficking.

Third, OMP can inhibit the fusion of phagosomes with lysosomes, which is essential for the survival and replication of L. pneumophila within the host cells. OMP interacts with Rab1 GTPase, a key regulator of vesicular transport, and prevents its recruitment to the Legionella-containing vacuole (LCV). OMP also interferes with the acidification of the LCV by blocking the activity of vacuolar ATPase (V-ATPase), a proton pump that maintains the pH gradient across the phagosomal membrane.

In summary, OMP is a multifunctional virulence factor that enables L. pneumophila to invade, manipulate, and persist in eukaryotic cells.

Another important virulence factor of L. pneumophila is the Mip protein, which belongs to the FK506-binding proteins (FKBP) family of peptidyl-prolyl cis/trans isomerases. Mip is essential for optimal intracellular survival and lung tissue dissemination of L. pneumophila . Mip also enhances the adherence and phagocytosis of L. pneumophila by macrophages . Mip interacts with collagen type I and IV on the surface of host cells and facilitates the bacterial entry through the CR3 complement receptor . Mip also modulates the host immune response by inducing the production of pro-inflammatory cytokines such as IL-6 and TNF-α. Mip is a potential drug target for treating Legionnaires` disease, as several small-molecule inhibitors have been identified that can block its enzymatic activity and impair its function . These inhibitors have shown promising results in reducing the bacterial load and inflammation in animal models of infection . Therefore, Mip is a crucial factor that contributes to the pathogenesis of L. pneumophila by promoting its attachment, uptake, and survival within host cells.

Type IV pili are thin, flexible, filamentous structures that protrude from the bacterial surface and mediate various functions such as adhesion, motility, biofilm formation, and DNA uptake . L. pneumophila expresses multiple types of pili with different lengths and compositions . One of these pili is encoded by a gene with homology to the type IV pilin genes (pilEL) . This pilin gene is expressed by L. pneumophila under certain environmental conditions and is required for optimal infection of macrophages and amoebas . The expression of this pilin gene also influences the trafficking of the L. pneumophila-containing vacuole (LCV) within the host cell . The LCV is a specialized membrane-bound compartment that allows L. pneumophila to evade lysosomal degradation and replicate intracellularly . The type IV pilin gene affects the recruitment of early endosomal markers such as Rab5 and EEA1 to the LCV, as well as the fusion of the LCV with other vesicles containing nutrients or bacterial effectors . The type IV pilin gene also modulates the interaction of the LCV with the microtubule network and the actin cytoskeleton, which are important for the movement and positioning of the LCV within the host cell . Thus, type IV pili play a role in the entry and intracellular survival of L. pneumophila by influencing the trafficking and maturation of the LCV.

Flagella are long, whip-like appendages that enable bacteria to swim and move. Legionella pneumophila has a single, polar flagellum that is important for its motility and virulence. Flagella can also act as adhesins, binding to host cell receptors and facilitating bacterial entry. Moreover, flagella can modulate the host immune response by stimulating toll-like receptor 5 (TLR5) and inducing pro-inflammatory cytokines.

Dot/icm type IV secretion system (T4SS) is a complex molecular machine that translocates bacterial effector proteins into the host cell cytosol. Dot/icm stands for defective organelle trafficking/intracellular multiplication, reflecting the essential role of this system in L. pneumophila pathogenesis. The Dot/icm T4SS consists of 27 proteins that form a membrane-spanning structure with a needle-like extension. The system recognizes and transports more than 150 different effectors that manipulate various host cell processes, such as vesicle trafficking, cytoskeleton dynamics, signal transduction, transcription, autophagy, and apoptosis.

One of the key functions of the Dot/icm T4SS is to create and maintain a specialized vacuole that supports bacterial replication inside the host cell. This vacuole, called the Legionella-containing vacuole (LCV), avoids fusion with lysosomes and acquires components from the endoplasmic reticulum (ER). The Dot/icm effectors modulate the LCV membrane and recruit ER-derived vesicles to provide nutrients and lipids for bacterial growth.

Another important function of the Dot/icm T4SS is to inhibit host cell apoptosis, or programmed cell death, which is a defense mechanism against intracellular pathogens. The Dot/icm effectors interfere with various apoptotic pathways, such as caspase activation, mitochondrial membrane potential, cytochrome c release, and Bcl-2 family proteins. By preventing apoptosis, L. pneumophila prolongs the survival of the host cell and allows more time for bacterial replication and dissemination.

In summary, flagella and Dot/icm T4SS are two major virulence factors of L. pneumophila that enable the bacterium to enter and survive within host cells. Flagella facilitate motility and adhesion, while Dot/icm T4SS delivers a large number of effectors that modulate host cell functions.

MOMPs are major outer membrane proteins of L. pneumophila that constitute about 75% of the total outer membrane proteins. They have multiple roles in the virulence and survival of the bacteria in different environments.

One of the functions of MOMPs is to mediate the adherence of L. pneumophila to various host cells, such as macrophages, epithelial cells, and protozoa. MOMPs bind to specific receptors on the host cell surface, such as integrins, laminin, and fibronectin, and facilitate the uptake of the bacteria by endocytosis or phagocytosis.

Another function of MOMPs is to promote the intracellular replication of L. pneumophila within the LCV. MOMPs interact with host cell proteins and modulate the signaling pathways that regulate the biogenesis and maturation of the LCV. For example, MOMPs activate the ERK1/2 pathway that inhibits the fusion of the LCV with lysosomes and enhances the recruitment of ER-derived vesicles to the LCV. MOMPs also induce the expression of anti-apoptotic genes that protect the infected host cell from death.

MOMPs also play a role in the biofilm development and formation of L. pneumophila on various surfaces, such as pipes, cooling towers, and water reservoirs. Biofilms are complex communities of microorganisms that adhere to each other and to a surface, forming a protective matrix of extracellular polymeric substances (EPS). Biofilms provide a favorable environment for L. pneumophila to persist, grow, and resist disinfection. MOMPs contribute to the biofilm formation by enhancing the attachment of L. pneumophila to abiotic surfaces and by regulating the production of EPS.

MOMPs also facilitate the horizontal gene transfer (HGT) of L. pneumophila with other bacteria in biofilms or in aquatic environments. HGT is the exchange of genetic material between different bacterial species or strains that can result in new traits or functions. MOMPs act as natural transformation mediators that bind to extracellular DNA and transport it into the bacterial cell. HGT can increase the genetic diversity and adaptability of L. pneumophila and may confer new virulence factors or antibiotic resistance genes.

In summary, MOMPs are multifunctional virulence factors of L. pneumophila that are involved in adherence, intracellular replication, biofilm development, and horizontal gene transfer. These functions enable L. pneumophila to infect and survive in various hosts and environments.

Legionella pneumophila is a gram-negative bacterium that can cause Legionnaires` disease, a severe form of pneumonia, or Pontiac fever, a mild flu-like illness. L. pneumophila is considered a facultative intracellular pathogen, meaning that it can survive and multiply both inside and outside host cells.

The main route of transmission of L. pneumophila is through inhalation of aerosolized water droplets contaminated with the bacteria. The bacteria can also enter the lungs by aspiration of drinking water. Once in the lungs, L. pneumophila attaches to the respiratory mucosa and invades alveolar macrophages, which are immune cells that normally phagocytose and kill pathogens. However, L. pneumophila has evolved several strategies to evade the host defense mechanisms and establish a replicative niche within the macrophages.

One of the key virulence factors of L. pneumophila is the Dot/Icm type IV secretion system (T4SS), which is a molecular syringe that injects more than 300 bacterial effector proteins into the host cell cytosol or membrane. These effectors manipulate various host cell processes, such as vesicle trafficking, cytoskeleton dynamics, signal transduction, apoptosis, autophagy, and innate immunity. The Dot/Icm T4SS enables L. pneumophila to avoid fusion of the phagosome with lysosomes, where the bacteria would be degraded by acidic and enzymatic conditions. Instead, L. pneumophila remodels the phagosome into a specialized vacuole called the Legionella-containing vacuole (LCV), which acquires nutrients and membrane components from the endoplasmic reticulum (ER) and other organelles. The LCV provides a favorable environment for bacterial replication and protects the bacteria from host immune responses.

Other virulence factors of L. pneumophila include surface structures such as type IV pili, flagella, and outer membrane proteins, which mediate bacterial adhesion, invasion, motility, and biofilm formation. L. pneumophila also produces a siderophore called legiobactin, which scavenges iron from the host cell and enhances bacterial growth. Moreover, L. pneumophila can modulate the expression of heat shock proteins (HSPs), such as HSP60 and HSP90, which enhance bacterial invasion and cytokine production in macrophages.

The intracellular replication of L. pneumophila leads to tissue damage and inflammation in the lungs. The infected macrophages secrete pro-inflammatory cytokines and chemokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1), which recruit other immune cells to the site of infection. However, some of the bacterial effectors can also dampen or subvert the host immune response by inhibiting cytokine secretion or signaling pathways. The balance between pro- and anti-inflammatory responses determines the outcome of infection.

The clearance of L. pneumophila from the lungs requires cell-mediated immunity, especially CD4+ T helper 1 (Th1) cells that produce interferon-γ (IFN-γ). IFN-γ activates macrophages to kill intracellular bacteria by enhancing phagosome-lysosome fusion and inducing nitric oxide production. Humoral immunity plays a minor role in protection against L. pneumophila infection.

In summary, L. pneumophila is a facultative intracellular pathogen that exploits macrophages as its primary host cells. The bacterium uses various virulence factors to evade host defense mechanisms and create a replicative niche within a specialized vacuole. The infection triggers an inflammatory response in the lungs that can be either beneficial or detrimental for the host. The elimination of L. pneumophila depends on cell-mediated immunity mediated by Th1 cells and IFN-γ.

Legionella pneumophila can cause two distinct clinical syndromes: Legionnaires` disease and Pontiac fever .

Legionnaires` disease

Legionnaires` disease is a severe form of pneumonia that can be fatal if untreated. It typically presents with fever, cough, shortness of breath, and chest pain, 2 to 10 days after exposure to contaminated water or soil . Other symptoms may include headache, body ache, chills, fatigue, gastrointestinal symptoms, and confusion . The infection is diagnosed by detecting the bacteria or its antigens in respiratory specimens or urine, or by serological tests . The treatment involves antibiotics, such as levofloxacin or azithromycin, and supportive care . The risk factors for acquiring Legionnaires` disease include advanced age, smoking, chronic lung disease, diabetes, kidney disease, cancer, and immunosuppression .

Pontiac fever

Pontiac fever is a milder, self-limiting illness that resembles influenza. It manifests with fever, chills, myalgia, malaise, and headache, 6 to 12 hours after exposure to contaminated water or soil . Respiratory symptoms are less common and mild in Pontiac fever than in Legionnaires` disease . The infection is diagnosed by serological tests or by epidemiological evidence of an outbreak . The treatment is supportive and no antibiotics are required . The attack rate of Pontiac fever is very high (>90%) and it affects healthy and young individuals as well as those with risk factors .

Legionella pneumophila infection can cause serious and potentially fatal pneumonia in humans. Therefore, timely and accurate diagnosis, treatment and prevention are essential to reduce morbidity and mortality.

The preferred diagnostic tests for Legionella pneumophila infection are culture of lower respiratory secretions (e.g., sputum, bronchoalveolar lavage) on selective media and the Legionella urinary antigen test. Serological assays can be nonspecific and are not recommended in most situations. Best practice is to obtain both sputum for culture and urine for the urinary antigen test concurrently. Sputum should ideally be obtained prior to antibiotic administration, but antibiotic treatment should not be delayed to facilitate this process. The urinary antigen test can detect Legionella infections in some cases for days to weeks after treatment.

Other diagnostic methods include direct fluorescent antibody (DFA) staining of respiratory specimens, polymerase chain reaction (PCR) assays, antigen detection in other body fluids (e.g., serum, cerebrospinal fluid), and histopathology with immunohistochemistry . However, these methods have limitations in sensitivity, specificity, availability or interpretation .

The treatment of Legionella pneumophila infection requires antibiotics that can penetrate the intracellular vacuole where the bacteria replicate. Either a fluoroquinolone (levofloxacin or moxifloxacin) or a macrolide (azithromycin preferred) is the recommended first-line therapy for Legionnaires’ disease . In severe cases, a fluoroquinolone or rifampicin can be added to a macrolide. The duration of treatment is usually 10 to 14 days, but may be longer in complicated cases or immunocompromised patients . Aminoglycoside and β-lactam antibiotics are not effective against Legionella pneumophila and should be avoided.

The prevention of Legionella pneumophila infection involves environmental control measures to reduce the risk of exposure to contaminated water sources. These include maintaining adequate water temperature and disinfection levels, avoiding stagnation and biofilm formation, cleaning and disinfecting cooling towers and evaporative condensers, and implementing routine monitoring and maintenance programs . In addition, high-risk groups such as travelers, healthcare workers and immunocompromised patients should be educated about the signs and symptoms of Legionnaires’ disease and seek medical attention promptly if they develop respiratory illness after exposure to potential sources of Legionella pneumophila .

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