Conclusion
The Dot Icm secretion system plays a crucial role in the generation of a replication-permissive niche for L. pneumophila in a wide range of protozoan and mammalian hosts. Although a limited amount of work has been done to determine how this T4SS assembles and functions, much labor remains. Recent estimates indicate that the Dot Icm system may export between 50 and 150 proteins making it one of the most robust bacterial secretion systems ever identified. Although the apparent functional...
Ylf and the Vips
Using a Saccharomyces cerevisiae model, Campodonico et al. 2005 demonstrated that over-production of L. pneumophila yeast lethal factor YlfA resulted in vesicular trafficking defects, an effect attributed to its N-terminal coiled-coil regions. By sequence analysis, a paralog to YflA, YflB, was identified. Both YlfA and YlfB were shown to be secreted into host cells by a Dot Icm-dependent process, although ylfA ylfB double mutants showed no intracellular growth defects in A. castellanii or...
References Stb
Amano K, Williams JC 1983. Partial characterization of peptidoglycan-associated proteins of Legionella pneumophila. J. Biochem Tokyo . 94 601-606. Aragon V, Kurtz S, Cianciotto NP 2001 . Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect. Immun. 69 177-185. Baker LM, Raudonikinene A, Hoffman PS, and Poole LB 2001 An essential thioredoxin-dependent peroxiredoxin from Helicobacter pylori genetic and kinetic characterization. J. Bacteriol. 183 1961-1973....
The Importance of Iron for Legionella pneumophila
Iron is a key requirement for L. pneumophila replication Reeves et al, 1981 . As originally defined, the iron requirement for L. pneumophila was estimated to be 3-13 M for minimal growth and gt 20 M for optimal growth Reeves et al, 1983 Johnson et al, 1991 Mengaud and Horwitz, 1993 . However, recent studies using chemically defined media indicate that the requirement is lt 1 M James et al, 1995 Liles et al, 2000 . In standard BCYE agar, iron is added in the form of ferric pyrophosphate,...
The Effects of EGCG on Costimulatory Molecule Expression by Legionellalnfected
A recent study concerning the effects of Lp infection of DCs showed that EGCG treatment inhibited surface expression of co-stimulatory molecules by BMDCs from Lp-resistant BALB c mice Fig. 10.2 . Expression of co-stimulatory molecules is a marker of DC maturation and associated with enhanced ability to activate resting T cells. Studies showed that EGCG treatment of cultured Lp-infected DCs inhibited the Lp-induced expression of both MHC class l and class ll molecules important for DC...
Stringent Response
Microbes trigger a global change in their cellular metabolism known as the stringent response when confronted with nutritional and metabolic stresses, such as the limitation of amino acids, carbon, nitrogen, or phosphate Magnusson et al. 2005 . Numerous physiological processes are coordinately altered, including growth inhibition, down-regulation of nucleic acid and protein synthesis, enhancement of protein degradation, and upregulation of amino acid synthesis and transport. The stringent...
Association with Biofilms
Interaction of legionellae and protozoa is impacted by other microorganisms that comprise biofilms in building water systems. Legionellae are known to colonize these biofilms and can persist within these microbial communities for years. They are more easily detected from swab samples of biofilm than from flowing water, suggesting that the majority of the legionellae are biofilm associated Rogers et al. 1994 . A limited number of studies have attempted to characterize the bacteria's interaction...
Natural Environments
A limited number of studies have looked at the distribution of legionellae in natural waters. Most studies have focused on man-made aquatic systems associated with disease transmission. Several early studies of natural environments relied heavily on direct fluorescent antibody staining for the detection of the bacteria in these samples. One study examined hundreds of samples from numerous lakes and rivers in the US Fleirmans et al. 1981 . This study detected legionellae in the majority of...
LepA and LepB
LepA and LepB were identified by sequence analysis the L. pneumophila genome for likely T4SS substrates and their export was confirmed to be Dot Icm dependent Chen et al., 2004 . Typical of other Dot Icm-substrates, inactivation of lepA, lepB, or both genes did not significantly diminish the intracellular replication of L. pneumophila within macrophages, although a lower survival rate was observed in a protozoan host Chen et al., 2004 . This phenotype appeared to be due to the non-lytic release...
Amino Acid Availability Governs Differentiation
Although the exact nutrient composition of the L. pneumophila replication vacuole is unknown, several lines of evidence indicate that amino acids are critical, and differences in these concentrations can affect the differentiation state of the microbe. Foremost, broth studies indicate that L. pneumophila depends on amino acids for its sole source of carbon and energy Tesh et al. 1983 . Additionally, the uptake of amino acids by its host cell via the human transporter protein SLC1A5 hATB0, is...
DotIcm Type IV Secretion System
At least two different T4SS have been identified in a number of L. pneumophila strains. The first T4SS, a type IVA secretion system, was designated Lvh for Legionella Vir homolog due to its similarities to the virB operon of A. tumefaciens. However, this system was shown to be dispensable for intra-cellular replication of L. pneumophila and is not present in several virulent strains Christie and Vogel, 2000 Segal et al., 1999 . The second T4SS, a type IVB secretion system, was named the Dot Icm...
Transcriptional Control of Differentiation via Sigma Factors
To regulate gene expression, one mechanism commonly employed by bacteria is to modify RNA polymerase RNAP activity by altering its sigma factor. In E. coli, RNAP consists of a core enzyme and one of seven potential sigma factors that direct the RNAP to a distinct cohort of promoters Nystrom 2004 . For growth-related activities and proliferation, E. coli require the housekeeping sigma factor, ct70, encoded by rpoD Nystrom 2004 . However, during conditions of growth arrest, starvation, stress, or...
Summary
Legionnaires' Disease which came to our attention in dramatic fashion in 1976 remains a most fascinating disease. It can occur in outbreaks or as sporadic cases both in the community and nosocomially. Transmission is through inhalation of contaminated aerosols or through aspiration of contaminated water. The methods of diagnosis and treatment have changed over the years. Now testing of urine for Legionella antigen is commonly used but culture of respiratory secretions and amplification of...
of L pneumophila
Similar to its intracellular fate within macrophages, L. pneumophila is enclosed, after entry into amoebae, in a phagosome surrounded by host cell organelles such as mitochondria, vesicles, and a multilayer membrane derived from the rough endoplasmic reticulum RER of amoeba Tilney et al., 2001 Abu Kwaik, 1996 Swanson and Isberg, 1995 Horwitz, 1983b . The Legionella-containing phagosome LCP evades endocytic fusion within mammalian and protozoan cells Abu Kwaik, 1996 Bozue and Johnson, 1996...
Respiratory Metabolism
Members of the genus Legionella exhibit a strictly respiratory form of metabolism and do not grow anaerobically. Moreover, anaerobic respiration with nitrate or other electron acceptors has not been demonstrated. The electron transport chain is composed of cytochromes of the b, c, aa3, and d types and the legionellae are cytochrome oxidase positive. The electron transport chain is branched with cytochrome c' aa3 cytochrome c oxidase complex and cytochrome d as terminal electron acceptors...
Innate Immunity to L pneumophila
The course of infection in susceptible experimental animals, such as genetically susceptible mice, can be divided into an early phase during which there is rapid bacterial multiplication and an inflammatory response. A second phase begins Figure 9.3. L. pneumophila infection stimulates cytokine production involved in both innate and acquired immune responses and initiates a cascade of immune factors. Figure 9.3. L. pneumophila infection stimulates cytokine production involved in both innate and...
PostTranscriptional Control of Differentiation 71 LetALetS TwoComponent System
Two-component systems are widely used by prokaryotic organisms to adapt to environmental fluctuations. Typically, these signal transduction systems consist of a membrane-bound sensor protein that monitors the environment, and a cyto-plasmic response regulator that binds target DNA sequences Appleby et al. 1996 Bijlsma and Groisman 2003 . Upon stimulation, the sensor autophosphorylates a conserved histidine residue using the 7-phosphoryl group of ATP as a donor Appleby et al. 1996 Bijlsma and...
Introduction
As a fundamental strategy for virulence, a large number of pathogens possess specialized secretion systems that are used to deliver effector proteins into host cells, thus modulating host cell function. In Gram-negative bacteria, these specialized secretion systems include type III secretion systems T3SSs Hueck, 1998 and type IV secretion systems T4SSs Backert and Meyer, 2006 Christie and Vogel, 2000 Christie et al., 2005 Sexton and Vogel, 2002 . T3SS components share similarities to flagellar...
Epidemiology
Legionnaires' disease may occur as sporadic cases or as outbreaks and in both instances the disease may be community or nosocomially acquired. It may also occur as a non-pneumonic form, Pontiac fever. In July 1968, an illness that involved a short incubation period, a high attack rate of 95 , with fever, headache, myalgia, and malaise. Just over half, 57 , had cough but no pneumonia occurred in 144 affected people in a new county health department building in Pontiac, Michigan Meyer 1983 Glick...
Morphology Ultrastructure and Surface Proteins
Legionella pneumophila displays a typical Gram-negative envelope in which the inner membrane and outer membrane can be readily distinguished in thin sections by electron microscopy see Figure 7.1a . In some thin sections of L. pneumophila, outer membrane blebs are observed, a feature typical of most Gram-negative bacteria. In contrast, the cyst form contains a thick cell wall laminations of intracytoplasmic membranes, and a cytoplasm occupied with inclusions of PHBA see Figure 7.1b,c,d . While...
Adaptive Immunity to L pneumophila Infection
The adaptive immune response to bacterial infection follows initial exposure to microbes, not necessarily active infection, and is transferable to naive recipients by lymphocytes. The humoral antibody response to bacterial infection is a typical adaptive immune response when an individual is exposed to bacterial antigens and a heightened specific antibody response then occurs after secondary stimulation by antigens from the same microbe or active infection. The role of antibodies in resistance...
The Sid Family
The Sid substrate of Icm Dot family of proteins were identified via a combination of a two-hybrid screen and a bacterial protein translocation assay Luo and Isberg, 2004 . In the original report one Sid protein, SidC, was localized to the cytoplasmic face of the LCV within macrophages Luo and Isberg, 2004 . Weber et al. 2006 recently reported that SidC and its paralog SdcA were able to specifically bind to phosphatidylinositol 4 phosphate PI 4 P found on the LCV, thus providing an explanation...
Heat Shock Response
One of the earliest stress responses studied in detail in L. pneumophila was the heat shock response which is common to all living organisms. Interest in heat shock proteins HSPs was initially driven by the finding of L. pneumophila in hot water systems, hot water tanks, and industrial boilers. In E. coli, the heat shock response is controlled by the heat shock sigma factor RpoH or ct32. Following heat shock, RpoH is activated and binds to promoter regions of genes containing heat shock...
Concluding Remarks 1
It seems clear now that L. pneumophila has a defined developmental cycle embedded in its life cycle. Developmental cycles are central to the pathogenesis and ecology of obligate intracellular bacterial pathogens with an extracellular phase. Therefore, differentiation into a highly infectious and environmentally resilient form that survives extended periods of starvation in freshwater just to have the opportunity to gain access into new hosts constitutes a trait evolved by L. pneu-mophila that...
References Hpv
1. Abu-Zant, A., M. Santic, M. Molmeret, S. Jones, J. Helbig, and Y. Abu Kwaik. 2005. Incomplete activation of macrophage apoptosis during intracellular replication of Legionella pneumophila. Infect Immun 73 5339-49. 2. Akamine, M., F. Higa, N. Arakaki, K. Kawakami, K. Takeda, S. Akira, and A. Saito. 2005. Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila. Infect Immun 73 352-61. 3. Andrews, H. L., J. P. Vogel, and R. R. Isberg....
AdaptorChaperone Complexes
L. pneumophila icmS and icmW mutants had a unique phenotype compared to other dot icm mutant strains since the LCV associated with vesicles, but then quickly fused with the endocytic pathway Coers et al., 2000 Zuckman et al., 1999 . IcmS and IcmW were both shown to bind to a number of Dot Icm-secreted proteins, and to interact with each other, suggesting that they function as an adaptor complex involved in substrate recognition by the Dot Icm complex Bardill et al., 2005 Coers et al., 2000...
The Effects of EGCG on Chemokine Production by Legionellalnfected Dendritic
Maturing DCs involved in innate or adaptive immunity are also an abundant source of chemokines in a precise time-ordered fashion. After stimulation with a microbial antigen, DCs have an initial burst of MlP-la CCL3 , MlP-1b CCL4 and lL-8 CXCL8 production within a few hours. RANTES CCL5 and MCP-1 are also induced but in a more steady manner. At a later time point DCs produce mainly lymphoid chemokines such as CCLl7 TARC , CCLl8 DC-CDl , CCLl9 MIP-3B and CCL22 MDC that attract T and B lymphocytes...
Antibacterial and Antiviral Effects of Epigallocatechin Gallate
The use of such substances to prevent or moderate infectious diseases is a cornerstone of traditional Chinese medicine and also alternative medicine in Western countries. In this regard, there has been increased interest in prophylactic treatment to prevent or ameliorate clinical infection or re-infection by opportunistic microbes which cause chronic disease, including gastritis due to H. pylori. Such substances are often derived from extracts of plants considered medicinal. In particular, many...
Structural Components
The Dot Icm system is a multiprotein structure that forms a channel spanning the bacterial inner and outer membranes Coers et al., 1999 . Although many of the Dot Icm components have not been extensively characterized, a number of them have now been examined revealing their localization and putative roles in the formation function of the secretion apparatus. The first to be examined, DotA, is a polytopic inner membrane protein that may act as a scaffolding protein Roy and Isberg, 1997 . DotB is...
Plasmids of Legionella
Two of the three sequenced L. pneumophila strains carry plasmids that might play a role either in adaptation to the environment or in virulence as both encode putative virulence factors, mobile genetic elements and antibiotic resistance genes. In strain Paris a 132 kb plasmid and in strain Lens a 60 kb plasmid was identified, both of which seem to be single copy plasmids. These plasmids contain a 24 kb Figure 1.5. Regions of plasticity identified in the L. pneumophila genomes and their...
Central Intermediary Metabolism
From whole genome analysis and from early biochemical and radiotracer studies we know that L. pneumophila contains a complete glycolytic pathway as well as key components of the pentose phosphate pathway and Entner Doudoroff pathway see Figure 7.2 . L. pneumophila lacks sugar transporters and must rely on gluconeogenesis for synthesis of sugars for biosynthesis of peptidoglycan, LPS, ribose, and deoxyribose and other cellular components. The bacteria can be grown in chemically defined medium...
Biosynthetic Capacity and Amino Acid Auxotrophies
Most species of Legionella can be grown in vitro on specialized yeast extract-based medium supplemented with ACES buffer, a-ketoglutarate, L-cysteine, and ferric pyrophosphate BCYEa medium Feeley et al., 1979 . This medium has not changed in 25 years. Early studies showed that charcoal and a-ketoglutarate function by quenching free radical reactions and thereby detoxified the medium of toxic reduced forms of oxygen Hoffman et al., 1983 . Several studies have further dissected the nutritional...
Entry of L pneumophila into Protozoa
The attachment and entry mechanisms of L. pneumophila into its protozoan hosts and variations in their mechanisms have been reported for both amoeba Hartmanella vermiformis and Acanthamoeba spp. Attachment of L. pneumophila to H. vermiformis is mediated by adherence to a protozoan receptor characterized as a putative galactose N-acetyl-galactosamine Gal GalNAc lectin Harb et al., 1998 Venkataraman et al., 1997 Mann et al., 1991 . Host protein synthesis by A. polyphaga is not required for...
Legionella pneumophila Development Along the Extracellular Growth Cycle
The extracellular growth cycle of L. pneumophila is also associated with a differentiation process, initially described by Byrne and Swanson 1998 . The post-exponential form or stationary phase form of L. pneumophila Fig. 4.2 Virulent form or post-exponential phase form Figure 4.3. The growth cycle of L. pneumophila in HeLa cells. The cycle begins with the attachment to and the invasion of a HeLa cell, a process mediated at least partially by the L. pneumophila chaperonin. Cell infection is...
Temporal Modulation of the LCV Surface
The identification of host pathways involved in LCV biogenesis may reveal which Dot Icm substrates are crucial for intracellular growth. Based on this assumption, Dorer et al. 2006 used RNA interference in Drosophila Kc167 cells to inhibit multiple host trafficking pathways that were important for intracellular growth of L. pneumophila. This approach identified Cdc48 p97, a component of endoplasmic reticulum-associated degradation ERAD , as playing an important role in the intracellular growth...
Motility and Chemotaxis
Several studies have shown that L. pneumophila is motile when grown at low temperature lt 30 C and non-motile during exponential phase at 37 C Dietrich et al., 2001 . However, as bacteria enter post-exponential growth, they become motile. This is unusual as most bacteria are motile during exponential phase of growth. During intracellular growth, bacteria become motile late in infection 37 C and motility is readily apparent within phagosomes of infected host cells cover slip assays of infected...
Pht Family of Transporters
To sense amino acid availability and determine if differentiation to a replicative state is advantageous, transmissive L. pneumophila use a family of phagosomal transporters Phts Sauer et al. 2005 . In particular, a L. pneumophila mutant of the phtA gene was shown to have a pronounced defect in intracellular growth in Figure 6.3. Pht transporters couple nutrient acquisition to microbial differentiation. Transport of amino acids by Pht proteins trigger intracellular transmissive L. pneumophila...
General Features and Organization of the L pneumophila Genomes
The complete genome sequences of three strains of L. pneumophila Paris, Lens and Philadelphia 1 are available at the time of writing Cazalet et al. 2004 Chien et al. 2004 . Legionella pneumophila has a single, circular chromosome, 3,503 610 base pairs bp Paris , 3,345 687 bp Lens and 3,397 754 bp Philadelphia 1 in size, with an average G C content of 38 Table 1.1 Figure 1.1. Strains Paris and Lens each contain a plasmid, 131.9 kb and 59.8 kb in size, respectively. In strain Philadelphia 1 no...
The Effects of EGCG on Cytokine Production by LegionellaInfected Dendritic Cells
Dendritic cells DCs are also phagocytic cells which play a vital role in innate immunity. However, the main characteristic of DCs is their potent ability to take up antigens, mature by undergoing various phenotypic and functional changes such as the upregulation of co-stimulatory molecules and the induction of an adaptive immune response through interaction with T cells. Unlike bone marrow derived macrophages BMMs , bone marrow derived dendritic cells BMDCs from susceptible A J mice are...
Genome Comparison of Legionella pneumophila and Coxiella Burnetii
Comparative genome analysis can be used to identify species-specific genes and gene clusters, and analysis of these genes can give insight into the mechanisms involved in specific host-pathogen interactions. We thus compared the genomes of L. pneumophila with that of the evolutionary most closely related species, C. burnetii. Coxiella burnetti is an obligate intracellular pathogen that, like Legionella, thrives on both macrophages and amoebae. Thus the comparison of these genomes should allow...
Legionella Pneumophila References
Aldridge P, Paul R, Goymer P, Rainey P, Jenal U 2003 Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol Microbiol 47 1695-708. Amer AO, Swanson MS 2005 Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol 7 765-778. Baar C, Eppinger M, Raddatz G, Simon J, Lanz C, Klimmek O, Nandakumar R, Gross R, Rosinus A, Keller H, Jagtap P, Linke B, Meyer F, Lederer H, Schuster SC 2003 Complete genome sequence and analysis of Wolinella...
Immunomodulatory Effects of Natural Products on TLR Expression and Signal
Toll-like receptors TLRs are an essential component for bacterial recognition and initiation of signaling pathways necessary for induction of cytokines, chemokines and co-stimulatory molecule expression in immune cells. Interestingly, there are several reports of inhibition of TLR induction by various medicinal plant-derived products such as ginsan in response to bacterial infection. Expression of TLRs, including TLR2, TLR4 and TLR9, as well as the adaptor molecule MyD88 was considerably...
Legionella Siderophores
It was reported in 1983 that L. pneumophila does not make siderophores Reeves et al, 1983 . This conclusion was based largely on results from Arnow and Csaky assays, which detect catecholate and hydroxamate structures, respectively. Years later, the issue of Legionella siderophores was revisited using the CAS assay, which detects iron chelators independently of structure Goldoni et al, 1991 . CAS reactivity was detected in statically grown L. pneumophila cultures, suggesting the existence of a...
The Life Cycle of L pneumophila as Currently Understood
Life cycles tell us a great deal about the natural history of a living organism. They are a synthesis of years of evolution, adaptation, specialization, and survival in nature. From an infectious disease standpoint, life cycles may provide clues to understand the mechanisms of pathogenesis of a particular parasite. The origin of pathogens and the evolution of virulence lie within the dominion of microbial ecology, and the life cycle of L. pneumophila surely is no exception. Amoebae are the...
Protection from Oxidative Stress
Bacteria living under aerobic conditions must deal with toxic effects of oxygen that can be generated exogenously or endogenously from oxidation reactions within the bacteria cell. Early studies established that L. pneumophila grew poorly on most laboratory medium and that the addition of starch or activated charcoal dramatically improved the ability of media to support good growth. L. pneumophila grows poorly on BCYEa medium when the charcoal is omitted. The charcoal was subsequently shown to...
References Jln
Abu Kwaik Y 1996 The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 62 2022-2028. Abu Kwaik Y, Gao L-Y, Harb OS, and Stone BJ 1997 Transcriptional regulation of the macrophage-induced gene gspA of Legionella pneumophila and phenotypic characterization of a null mutant. Mol Microbiol 24 629-642. Abu Kwaik Y, Gao L-Y, Stone BJ, Venkataraman C, and Harb OS 1998 Invasion of protozoa...
Ralf Legionella
RalF, the recruitment of ARF to Legionella-phagosome factor, was the first identified native Dot Icm substrate Nagai et al., 2002 . RalF contains a Sec7 domain, which functions as a guanine nucleotide exchange factor GEF for the Arf family of GTPases Amor et al., 2005 Nagai et al., 2002 . Dot Icm-translocated RalF recruits and activates the host GTPase ADP-ribosylation factor Arf -1 to the LCV Nagai et al., 2002 . Since Arf1 is a well-characterized regulator of vesicular traffic, it was...
Clinical Features of Legionnaires Disease
A listing of symptoms and signs in patients with LD does not accurately convey the clinical features of the disease. While, as will be subsequently described, there are no features of LD that allow one to make the diagnosis clinically, there is little doubt from clinical observations that in young adults with no comorbidity who develop rapidly progressive pneumonia LD has to be near the top of the differential diagnosis. In the Philadelphia outbreak Tsai et al 1979 fever was present in 97...
RpoS
The stationary phase sigma factor, RpoS, is required for sodium sensitivity, maximal expression of flagellin, and lysosomal evasion, but it is dispensable for the other known transmissive phase phenotypes Hales and Shuman 1999 Bachman and Swanson 2001 . In accordance with the theory of sigma factor competition, over-expression of rpoS decreases csrA, letE, fliA, and flaA transcripts, and inhibits the fliA-dependent transmission traits of motility, infectivity, and cytotoxicity Bachman and...
Putative Virulence Factors as Deduced from Sequence Analysis
In addition to the eukaryotic-like proteins, proteins with homology to virulence factors of other organisms were identified through sequence analysis. The three sequenced L. pneumophila strains encode a protein homologous to the enteropath-ogenic E. coli EPEC virulence regulator BipA. These three Legionella proteins lpp2875, lpl2737, lpg2822 are identical and show 68 similarity to the E. coli BipA protein. The Legionella bipA gene has 43 GC content, which is significantly higher than that of...