No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The Legionella pneumophila icm locus: A set of genes required for intracellular multiplication in human macrophages
Abstract
Legionella pneumophila, the causative agent of Legionnaires' disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular life-style is the ability of the organism to replicate within a specialized phagosome which does not fuse with lysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome-lysosome fusion. In a previous study, a 12 kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-lysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-lysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1-2.4 kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.
... Allows for growth in the cell. LCV [75][76][77] PI4P Localization of effectors to LCV early during infection. [48,78] PmrA Allows for intracellular growth in host cells. ...
... Allows for growth in the cell and genetic conjugation. LCV [75][76][77]111,112] ankB Intracellular replication in U937, A. polyphaga, and human monocyte-derived macrophages. [62] ccm locus Growth, intracellular infection and virulence, especially in low iron environments Cytochrome c [25] dotA Intracellular replication within macrophages, required in the immediate stages of infection to prevent lysozyme fusion. ...
... Not required for growth within amoeba or nematodes. [75,[113][114][115][116][117] dotB Not required for intracellular growth in amoeba. [113] dotO Infection of A549 alveolar epithelial cells. ...
Legionella pneumophila (L. pneumophila) is a pathogenic bacterium of increasing concern, due to its ability to cause a severe pneumonia, Legionnaires’ Disease (LD), and the challenges in controlling the bacteria within premise plumbing systems. L. pneumophila can thrive within the biofilm of premise plumbing systems, utilizing protozoan hosts for protection from environmental stressors and to increase its growth rate, which increases the bacteria’s infectivity to human host cells. Typical disinfectant techniques have proven to be inadequate in controlling L. pneumophila in the premise plumbing system, exposing users to LD risks. As the bacteria have limited infectivity to human macrophages without replicating within a host protozoan cell, the replication within, and egress from, a protozoan host cell is an integral part of the bacteria’s lifecycle. While there is a great deal of information regarding how L. pneumophila interacts with protozoa, the ability to use this data in a model to attempt to predict a concentration of L. pneumophila in a water system is not known. This systematic review summarizes the information in the literature regarding L. pneumophila’s growth within and egress from the host cell, summarizes the genes which affect these processes, and calculates how oxidative stress can downregulate those genes.
... Type IV Secretion System Genome sequencing revealed that R. prowazekii contains the genes virB4, virB8, virB9, virB10, virB11 and virD4 (Andersson et al., 1998), which are homologues of the type IV secretion systems founded in Agrobacterium tumefaciens (virB operon; Stachel and Nester, 1986;Kuldau et al., 1990), Brucella abortus (virB; Sieira et al., 2000), E. coli (tra genes; Winans and Walker, 1985;Pohlman et al., 1994), Bordetella pertussis (ptl genes; Weiss et al., 1993;Kotob et al., 1995), Legionella pneumophila (dot-icm genes and lvh genes; Brand et al., 1994;Segal and Shuman, 1997;Segal et al., 1999), Helicobacter pylori (cag genes; Covacci et al., 1999), E. chaffeensis, A. phagocytophilum (Ohashi et al., 2002), and Wolbachia (Brand et al., 1994;Masui et al., 2000;Sieira et al., 2000). The type IV secretion systems inject either bacterial DNA or proteins directly into the cytosol of eukaryotic host cells (Winans and Walker, 1985;Weiss et al., 1993;Christie, 1997;Covacci et al., 1999). ...
... Type IV Secretion System Genome sequencing revealed that R. prowazekii contains the genes virB4, virB8, virB9, virB10, virB11 and virD4 (Andersson et al., 1998), which are homologues of the type IV secretion systems founded in Agrobacterium tumefaciens (virB operon; Stachel and Nester, 1986;Kuldau et al., 1990), Brucella abortus (virB; Sieira et al., 2000), E. coli (tra genes; Winans and Walker, 1985;Pohlman et al., 1994), Bordetella pertussis (ptl genes; Weiss et al., 1993;Kotob et al., 1995), Legionella pneumophila (dot-icm genes and lvh genes; Brand et al., 1994;Segal and Shuman, 1997;Segal et al., 1999), Helicobacter pylori (cag genes; Covacci et al., 1999), E. chaffeensis, A. phagocytophilum (Ohashi et al., 2002), and Wolbachia (Brand et al., 1994;Masui et al., 2000;Sieira et al., 2000). The type IV secretion systems inject either bacterial DNA or proteins directly into the cytosol of eukaryotic host cells (Winans and Walker, 1985;Weiss et al., 1993;Christie, 1997;Covacci et al., 1999). ...
... The type IV secretion systems inject either bacterial DNA or proteins directly into the cytosol of eukaryotic host cells (Winans and Walker, 1985;Weiss et al., 1993;Christie, 1997;Covacci et al., 1999). In the facultative intracellular bacteria L. pneumophila and B. abortus, the type IV secretion systems are essential for the intracellular multiplication of the two organisms (Brand et al., 1994;Sieira et al., 2000). Legionella pneumophila icm mutant is deficient in its ability to cause lysis of the host cell. ...
IntroductionRickettsiae are genetically related α-Proteobacteria with fascinating obligately intracellular lifestyles usually involving alternation between vertebrate and invertebrate hosts. These feats are accomplished with small, evolutionarily selected genomes. The agents classified as Rickettsia, Orientia, Ehrlichia, Anaplasma, Wolbachia and Neorickettsia cause diseases (such as epidemic louse-borne typhus fever, Rocky Mountain spotted fever, scrub typhus, human ehrlichioses, and bovine anaplasmosis) that have repeatedly altered the course of history (Zinsser, 1935), unexpectedly kill previously healthy tick-exposed persons, occur as highly prevalent endemic febrile illness, and are veterinary pathogens of major economic importance.Although mostly known as pathogens transmitted by hematophagous ticks and insects, some of the Rickettsiales are insect endosymbionts (Wolbachia) that have evolved dramatic abilities to manipulate their host populations, and others (Neoricket ...
... Upon uptake by macrophages, L. pneumophila resides within a specialized compartment termed the L. pneumophila-containing vacuole (LCV) (Horwitz, 1983). However, in order to survive and replicate within the LCV, L. pneumophila relies on its Dot/Icm type IVb secretion system (T4SS) (Berger & Isberg, 1993;Berger, Merriam, & Isberg, 1994;Brand, Sadosky, & Shuman, 1994;Ensminger & Isberg, 2009;Roy & Isberg, 1997). The T4SS translocates over 300 bacterial effectors that are essential for manipulation of host cell processes as well as for vacuolar remodeling (Hubber & Roy, 2010). ...
... pneumophila is a gram-negative intracellular bacterial pathogen that infects alveolar macrophages and is the causative agent of the severe pneumonia known as Legionnaires' Disease (Fraser et al., 1977). Upon uptake, L. pneumophila resides within a L. pneumophila-containing vacuole (LCV) and relies on the Dot/Icm type IV secretion system (T4SS) to survive within the LCV (Berger & Isberg, 1993;Berger et al., 1994;Brand et al., 1994;Horwitz, 1983;X. Liu & Shin, 2019;Roy & Isberg, 1997). ...
Host recognition of intracellular bacterial pathogens results in the formation of a multiprotein complex termed the inflammasome, which leads to the recruitment and activation of inflammatory caspases. These caspases promote IL-1 family cytokine release and pyroptosis, a lytic form of cell death, which are critical for anti-bacterial defense. In mice, interferon-gamma (IFN-γ) is a potent inducer of the both the noncanonical and canonical inflammasomes. Specifically, a family of IFN-inducible GTPases known as guanylate binding proteins (GBPs) promote inflammasome responses to a variety of bacteria in mice. The functions of the mouse GBPs include stimulating the rupture of pathogen-containing vacuoles and bacteriolysis of cytosolic bacteria in order to release pathogen-derived products, such lipopolysaccharide (LPS), into the cytosol for downstream host sensing and inflammasome activation. In contrast to mice, which possess 11 GBPs, humans have 7 GBPs and their role in inflammasome activation in human macrophages is poorly understood. Here, we use Legionella pneumophila, a vacuolar intracellular gram-negative bacterium, to elucidate the functions of human GBPs on inflammasome responses in macrophages. We determined that human GBP1 is essential for maximal inflammasome responses to L. pneumophila as well as is important for disrupting the L. pneumophila-containing vacuole (LCV) in order to make this vacuolar bacterium more accessible for cytosolic sensing. In addition, LPS can possess different acylation states within the same or different species of gram-negative bacteria. The noncanonical inflammasome mediates inflammatory responses to intracellular LPS and is comprised of caspase-11 in mice, and the two putative orthologs in humans, caspase-4 and caspase-5. While tetra-acylated LPS variants evade caspase-11 detection, caspase-4 was found to be activated by a tetra-acylated LPS variant. However, it is still unclear whether caspase-4 and caspase-5 recognize different LPS variants and whether their activation is promoted by IFN-γ. Here, we use primary or THP-1-derived macrophages and CRISPR/Cas9 technology to test human noncanonical inflammasome responses to LPS variants from different bacteria and whether IFN-γ and human GBPs promote these responses in macrophages. Our findings elucidate aspects of human innate immune response to gram-negative bacterial pathogens and may provide insight into developing therapeutics to prevent gram-negative sepsis.
... L. pneumophila replication in macrophages depends on the type IVb secretion system Dot/Icm 10,11 , which translocates over 300 L. pneumophila proteins into the host cell 12-14 . While the Dot/ Icm system is absolutely required for virulence 15,16 , the absence of individual Icm/Dot translocated substrates (IDTS) rarely impacts intracellular growth 17 , which is largely due to redundancy among these proteins 18,19 . For example, a L. pneumophila mutant lacking 31% of its IDTS is not defective for growth in cultured macrophages 20 . ...
... Eighty-seven genes were required for growth in all amoebae examined ( Fig. 1b and 15,16 . The impact of mutations in most genes, however, was host-specific, with 145 genes required in a subset of amoebae and 177 genes required for growth in only a single host (Fig. 1b). ...
Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.
... With Falkow's molecular Koch's postulates in mind, several genetic screens to correlate gene disruptions with virulence defects have been employed to identify L. pneumophila virulence genes (Berger and Isberg, 1993;Sadosky et al., 1993;VanRheenen et al., 2004;Laguna et al., 2006). Parallel genetic screens independently identified a collection of 26 genes encoding components of a Type IVb secretion system, subsequently named Icm/Dot (Marra et al., 1992;Berger and Isberg, 1993;Brand et al., 1994). Mutations in icm/dot genes abolish L. pneumophila intracellular growth in macrophages (Berger and Isberg, 1993;Brand et al., 1994) and amoebal hosts (Segal and Shuman, 1999) demonstrating a critical role for the Icm/Dot complex in L. pneumophila pathogenesis. ...
... Parallel genetic screens independently identified a collection of 26 genes encoding components of a Type IVb secretion system, subsequently named Icm/Dot (Marra et al., 1992;Berger and Isberg, 1993;Brand et al., 1994). Mutations in icm/dot genes abolish L. pneumophila intracellular growth in macrophages (Berger and Isberg, 1993;Brand et al., 1994) and amoebal hosts (Segal and Shuman, 1999) demonstrating a critical role for the Icm/Dot complex in L. pneumophila pathogenesis. The identification of Icm/Dot was not surprising as numerous pathogens employ secretion systems to deploy proteins, termed effectors to the host cell to establish growth. ...
Redundancy has been referred to as a state of no longer being needed or useful. Microbiologists often theorize that the only case of true redundancy in a haploid organism would be a recent gene duplication event, prior to divergence through selective pressure. However, a growing number of examples exist where an organism encodes two genes that appear to perform the same function. For example, many pathogens translocate multiple effector proteins into hosts. While disruption of individual effector genes does not result in a discernable phenotype, deleting genes in combination impairs pathogenesis: this has been described as redundancy. In many cases, this apparent redundancy could be due to limitations of laboratory models of pathogenesis that do not fully recapitulate the disease process. Alternatively, it is possible that the selective advantage achieved by this perceived redundancy is too subtle to be measured in the laboratory. Moreover, there are numerous possibilities for different types of redundancy. The most common and recognized form of redundancy is functional redundancy whereby two proteins have similar biochemical activities and substrate specificities allowing each one to compensate in the absence of the other. However, redundancy can also exist between seemingly unrelated proteins that manipulate the same or complementary host cell pathways. In this article, we outline 5 types of redundancy in pathogenesis: molecular, target, pathway, cellular process, and system redundancy that incorporate the biochemical activities, the host target specificities and the impact of effector function on the pathways and cellular process they modulate. For each type of redundancy, we provide examples from Legionella pathogenesis as this organism employs over 300 secreted virulence proteins and loss of individual proteins rarely impacts intracellular growth. We also discuss selective pressures that drive the maintenance of redundant mechanisms, the current methods used to resolve redundancy and features that distinguish between redundant and non-redundant virulence mechanisms.
... In spite of their intracellular lifestyle, early studies established culture conditions to allow growth of L. pneumophila in a free-living form [2], which enabled researchers to take advantage of genetic analysis to dissect the molecular mechanisms of L. pneumophila pathogenesis. By the late nineties, genetic analyses of L. pneumophila revealed that 30 genes are required for intracellular replication and/or cytotoxicity in eukaryotic host cells [3][4][5][6][7][8][9][10][11]. These genes were named dot (defect in organelle trafficking) or icm (intracellular multiplication) by Ralph Isberg's and Howard Shuman's groups, respectively. ...
... The Dot/Icm T4BSS includes two cytosolic small acidic proteins IcmS and IcmW, the properties of which resemble that of transport chaperones of type III secretion systems [6,10,37,38]. In bacterial cells IcmS and IcmW interacts with each other, and form complexes containing a substrate protein [39][40][41]. ...
... T4BSS are typically represented by the effector translocator Dot/Icm system from intracellular pathogens such as Legionella pneumophila and Coxiella burnetii (Berger and Isberg, 1993;Brand et al., 1994;Segal and Shuman, 1997). However T4BSS also include conjugative system members from the plasmid incompatibility group I such as the R64 plasmid from Salmonella enterica serovar Typhimurium and the ColIb-P9 plasmid from Shigella flexneri (Hartskeerl et al., 1985;Sampei et al., 2010) and recently a bacterial killing system from Pseudomonas putida (Purtschert-Montenegro et al., 2022). ...
Yersinia pseudotuberculosis is a food-borne pathogen responsible for a self-limiting gastrointestinal disease in humans known as mesenteric lymphadenitis. A phylogenetically distinct Y. pseudotuberculosis cluster from lineages 1 and 8 is associated to a specific syndrome called the Far East scarlet-like fever (FESLF), characterized by skin rash, hyperemic tongue and desquamation. Genome sequencing of FESLF strains previously revealed the presence in the plasmid pVM82 of dot/icm genes, homologous to those known to encode a T4BSS in the intracellular pathogens Legionella pneumophila and Coxiella burnetii. In the present article, we characterized the genomic features and functionality of the Y. pseudotuberculosis T4BSS ( y T4BSS). We found higher dot/icm gene identity between Y. pseudotuberculosis and Pseudomonas putida genes than with those of L. pneumophila or C. burnetii . We validated the presence of all essential dot/icm genes required for the structure of a T4BSS. We then evaluated the conditions required for y T4BSS gene expression in vitro and identified an influence of temperature, with higher expression at 37°C, which mimicks the mammalian host temperature. The y T4BSS is also expressed in cellulo during the Y. pseudotuberculosis intracellular life cycle and in vivo during mouse infection. Although T4BSS functions are well characterized in the intracellular life cycle of L. pneumophila and C. burnetii , the y T4BSS appears to not be required for the intracellular survival nor for the establishment of a replication niche within cells of Y. pseudotuberculosis . Interestingly, the y T4BSS is implicated in Y. pseudotuberculosis FESLF strain pathogenicity when orally inoculated to mice but not during intravenous inoculation. Despite a role in virulence during oral infection, the y T4BSS does not influence organ colonization. However, the y T4BSS appears to be implicated in induction of important necrosis lesions in mesenteric lymph nodes and cæca of mice. Cytokine profil analyses revealed an induction of production of innate immunity related cytokines and chemokines depending on the y T4BSS in cellulo using a mouse bone marrow-derived macrophages infection model. Thus, the y T4BSS modulates cytokine responses of the host innate immune system during oral infection. In conclusion, the y T4BSS is a newly characterized virulence factor implicated in pathogenicity of Y. pseudotuberculosis strains from lineage 8 responsible for FESLF.
... L egionella species are ubiquitous in the environment and have adapted to survive within freshwater amoebas (1,2). The bacteria rely on a type IV secretion system (T4SS) named defective in organelle trafficking and intracellular multiplication (Dot/Icm) to inject proteins into the host cell to manipulate host response pathways and increase their chances of survival (3)(4)(5)(6). Legionella can infect a wide variety of amoeba species and therefore express multiple homologs of bacterial effectors to successfully modify the same host cell target in various hosts (7). Humans are accidental hosts that can be exposed to Legionella via the aerosolization of contaminated water supplies, which may result in respiratory infections (8). ...
The host type I interferon (IFN) response protects against Legionella pneumophila infections. Other bacterial pathogens inhibit type I IFN-mediated cell signaling; however, the interaction between this signaling pathway and L. pneumophila has not been well described. Here, we demonstrate that L. pneumophila inhibits the IFN-β signaling pathway but does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-β to L. pneumophila -infected macrophages limited bacterial growth independently of NOS2 and reactive nitrogen species. The type IV secretion system of L. pneumophila is required to inhibit IFN-β-mediated cell signaling. Finally, we show that the inhibition of the IFN-β signaling pathway occurs downstream of STAT1 and STAT2 phosphorylation. In conclusion, our findings describe a novel host cell signaling pathway inhibited by L. pneumophila via its type IV secretion system.
... L. pneumophila is a Gram-negative pathogen that is the causative agent of the severe pneumonia Legionnaires' disease (36,37). Upon uptake into macrophages, this pathogen relies on the Dot/Icm type IV secretion system (T4SS) to replicate within an L. pneumophila-containing vacuole (LCV) (38)(39)(40)(41). The T4SS injects over 300 effector proteins, many of which enable L. pneumophila to evade the endolyso somal pathway and modify its LCV into an ER-derived replicative compartment (42)(43)(44)(45). ...
The inflammasome is essential for host defense against intracellular bacterial pathogens, including Legionella pneumophila , the causative agent of the severe pneumonia Legionnaires’ disease. Inflammasomes recruit and activate caspases, which promote IL-1 family cytokine release and pyroptosis to restrict infection. In mice, interferon (IFN) signaling promotes inflammasome responses against L. pneumophila and other bacteria , in part, through inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the L. pneumophila -containing vacuole (LCV), while GBPs are dispensable for vacuole rupture. Instead, GBPs facilitate the lysis of cytosol-exposed L. pneumophila . In contrast, the functions of IFN-γ and GBPs in human inflammasome responses to L. pneumophila are poorly understood. Here, we show that IFN-γ enhances caspase-1- and caspase-4-dependent inflammasome responses to L. pneumophila in human macrophages. We find that human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, we find that GBP1 co-localizes with L. pneumophila and/or LCVs in a type IV secretion system (T4SS)-dependent manner and facilitates damage to the LCV, resulting in increased bacterial access to the host cell cytosol. Our findings reveal species- and pathogen-specific differences in how GBPs function during infection.
IMPORTANCE
Inflammasomes are essential for host defense against intracellular bacterial pathogens like Legionella , as they activate caspases, which promote cytokine release and cell death to control infection. In mice, interferon (IFN) signaling promotes inflammasome responses against bacteria by inducing a family of IFN-inducible GTPases known as guanylate-binding proteins (GBPs). Within murine macrophages, IFN promotes the rupture of the Legionella -containing vacuole (LCV), while GBPs are dispensable for this process. Instead, GBPs facilitate the lysis of cytosol-exposed Legionella . In contrast, the functions of IFN and GBPs in human inflammasome responses to Legionella are poorly understood. We show that IFN-γ enhances inflammasome responses to Legionella in human macrophages. Human GBP1 is required for these IFN-γ-driven inflammasome responses. Furthermore, GBP1 co-localizes with Legionella and/or LCVs in a type IV secretion system (T4SS)-dependent manner and promotes damage to the LCV, which leads to increased exposure of the bacteria to the host cell cytosol. Thus, our findings reveal species- and pathogen-specific differences in how GBPs function to promote inflammasome responses.
... The Dot/Icm complex localizes to the bacterial poles (50,51), and its mis-localization impairs intracellular replication (50). Bacteria with defects in Dot/Icm function fail to remodel their phagosomes into replication compartments and instead traffic along the endocytic pathway to the lysosome (33,(52)(53)(54). ...
Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.
... The T4SS translocates over 330 effector proteins into the host cell. These factors interact with the host cell in many different ways to foster bacterial replication [35]. Despite this large array of factors, epigenetic regulation of host gene expression has only been shown in the case of RomA and hereby Smh1. ...
Legionella pneumophila (L.p.) is a bacterial pathogen which is a common causative agent of pneumonia. In humans, it infects alveolar macrophages and transfers hundreds of virulence factors that interfere with cellular signalling pathways and the transcriptomic landscape to sustain its own replication. By this interaction, it has acquired eukaryote-like protein motifs by gene transfer events that partake in the pathogenicity of Legionella. In a computational screening approach for eukaryotic motifs in the transcriptome of Legionella, we identified the L.p. strain Corby protein ABQ55614 as putative histone-deacetylase and named it “suppressing modifier of histones 1” (Smh1). During infection, Smh1 is translocated from the Legionella vacuole into the host cytosol. When expressed in human macrophage THP-1 cells, Smh1 was localized predominantly in the nucleus, leading to broad histone H3 and H4 deacetylation, blunted expression of a large number of genes (e.g. IL-1β and IL-8), and fostered intracellular bacterial replication. L.p. with a Smh1 knockdown grew normally in media but showed a slight growth defect inside the host cell. Furthermore, Smh1 showed a very potent histone deacetylation activity in vitro, e.g. at H3K14, that could be inhibited by targeted mutation of the putative catalytic center inferred by analogy with eukaryotic HDAC8, and with the deacetylase inhibitor trichostatin A. In summary, Smh1 displays functional homology with class I/II type HDACs. We identified Smh1 as a new Legionella virulence factor with a eukaryote-like histone-deacetylase activity that moderates host gene expression and might pave the way for further histone modifications.
IMPORTANCE
Legionella pneumophila (L.p.) is a prominent bacterial pathogen, which is a common causative agent of pneumonia. In order to survive inside the host cell, the human macrophage, it profoundly interacts with host cell processes to advance its own replication. In this study, we identify a bacterial factor, Smh1, with yet unknown function as a host histone deacetylase. The activity of this factor in the host cell leads to attenuated gene expression and increased intracellular bacterial replication.
... Instead, the bacterium acquires proteins and membranes from the early secretory pathway and other sources to establish a camouflaged replication compartment (22). L. pneumophila mutants with a non-functional T4SS fail to control trafficking of their LCV and are quickly delivered to lysosomes for degradation (23)(24)(25), thus underscoring the importance of the Dot/Icm system for Legionella pathogenesis. ...
Multi-drug-resistant pathogens are an emerging threat to human health. Because conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications.
... Instead, they help create a facultative niche for the pathogen's survival by functioning in concert with each other and subtly manipulating critical cellular pathways of the infected eukaryote (5,6). Pathogenic bacteria like Legionella pneumophila exploit eukaryotic cell functions and influence multiple signaling events by translocating over 330 effectors via its Type IV secretion system, also known as the Icm/Dot transporter (7)(8)(9)(10)(11). These effectors, collectively termed Icm/Dot translocated substrates (IDTS), either remain cytosolic, localize to the LCV or traffic to different organelles. ...
Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.
... Bien que les amibes et les macrophages humains soient les modèles cellulaires classiques pour l'étude de la virulence de L. pneumophila, il apparait que L. pneumophila est capable de coloniser d'autres organismes modèles tels que des lignées de cellules non phagocytaires de type HeLa (Garduño et al., 1998b), les souris permissives A/J et les lignées cellulaires qui en dérivent (Brieland et al., 1994), le nématode Caenorhabditis elegans (Brassinga et al., 2010), la fausse teigne de la cire Galleria melonella (Harding et al., 2012) ou encore la drosophile Drosophila melanogaster (Sun et al., 2013). Le fait que L. pneumophila soit capable de se multiplier dans des cellules phagocytaires très différentes d'un point de vue évolutif (des protistes unicellulaires aux macrophages de mammifère) révèle toute l'adresse de cette bactérie à contrôler des voix cellulaires conservées au sein de ses hôtes eucaryotes (Segal & Shuman, 1999 -Weissenberger et al., 2007;Berger et al., 1994;Brand et al., 1994;Segal & Shuman, 1997;Vogel et al., 1998). Bien que les protéines de ce système soient encore peu connues au niveau fonctionnel, on peut les repartir en six catégories. ...
Legionella pneumophila est une bactérie opportuniste qui émerge de l'environnement après multiplication dans des amibes et peut infecter accidentellement les macrophages alvéolaires humains, provoquant une pneumonie sévère, la légionellose. La capacité de L. pneumophila à survivre dans ses cellules hôtes est strictement dépendante du système de sécrétion de type 4 Dot/Icm, qui sécrète un large répertoire d'effecteurs dans le cytosol de l'hôte. Identifier la contribution individuelle de chaque protéine bactérienne sécrétée par le système Dot/Icm, dans le cycle infectieux de L. pneumophila reste un enjeu majeur pour comprendre les bases moléculaires de la virulence des légionelles. Mes travaux de thèse participent à cet objectif en caractérisant la voie cellulaire ciblée par la protéine kinase LegK2. Des tests d'interaction et de phosphorylation ont identifié le complexe nucléateur d'actine ARP2/3 comme cible de LegK2. Suite à l'adressage de LegK2 à la surface de la vacuole après sa translocation dans le cytosol de l'hôte, l'interaction LegK2-ARP2/3 inhibe la polymérisation d'actine sur le phagosome. Cette inhibition permet à Legionella de diminuer le trafic des endosomes tardifs et/ou des lysosomes vers le phagosome et favorise ainsi l'évasion du phagosome à la voie de dégradation endocytique. L'interaction LegK2-ARP2/3 met en évidence un mécanisme original de virulence dans lequel le remodelage local du cytosquelette d'actine de la cellule hôte permet à la bactérie de manipuler le trafic vésiculaire pour échapper aux défenses de l'hôte
... So far, it has been shown that ~300 substrates are transported by L. pneumophila into the host cells cytosol via the Dot/Icm system . This T4BSS is essential to Legionella virulence as dot/icm deficient mutant are unable to create proper LCV and to proliferate within amoebae or macrophages (Brand et al., 1994). ...
Legionella pneumophila is the causative agent of a form of pneumonia called legionellosis or Legionnaires’ disease. Between 2012 and 2015, the reported European cases of legionellosis increased from 5,848 to 7,069 cases per year where France, Germany, Italy and Spain accounted for 69% of the reported cases. Worryingly, the case fatality of incidents was 8.2% making this disease a considerable health concern. One virulence factor produced by this bacterium is a large protein (~700 kDa) belonging to the RTX (Repeats in ToXin) family called RtxA secreted by the type 1 secretion system. The hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (a.a. 108-109). We also show using lapG and lapD mutant strains, that RtxA release is controlled by these two proteins similar to Pseudomonas fluorescenes LapA. We observed that a strain lacking LapG protease maintains RtxA on the cell surface, while a strain lacking LapD does not exhibit cell surface RtxA. Interestingly, we identified the presence of homologous potential T1SS/LapDG systems in many Legionella species and other Gammaproteobacteria. Regarding L. pneumophila virulence, our work showed that mutants for L. pneumophila T1SS (lssBD/tolC) were more disruptive to its virulence than lapG/lapD mutants. We also hypothesize, by challenging infection, that L. pneumophila might be actively targeting its host via RtxA. Additionally, by observing rtxA mutants as well as detecting RtxA on host surface briefly after inoculation and attenuating virulence by using anti RtxA antibodies, we assume an important but not limiting role for this protein in the infection process
... This type of protein secretion system involves three steps: (i) recognition of effector proteins by a coupling protein complex, (ii) unfolding of the effector proteins in the cytoplasm, and (iii) translocation of the effector proteins in an unfolded state through a transenvelope conduit [4][5][6][7] . L. pneumophila has a Dot/Icm type IVB secretion system (T4BSS) [7][8][9] , which is composed of 26 Dot/ Icm (defective for organelle trafficking/intracellular multiplication defect) proteins and the adaptor protein LvgA [10][11][12][13][14] . It is made up of two major complexes, the core transenvelope conduit and a Dot/Icm type IV coupling protein (T4CP) complex. ...
The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.
... The T4BSS transporters evolved from I-type conjugation systems. The representative L. pneumophila Dot/Icm system, so named because the Isberg and Shuman groups independently discovered it and respectively named it Dot and Icm (defective in organelle trafficking/intracellular multiplication system), requires over >25 proteins of which only a few are VirB homologs (Marra et al. 1992;Sadosky et al. 1993;Berger and Isberg 1993;Brand et al. 1994;Nagai and Kubori 2011;Voth et al. 2012). The T4ASS and T4BSS classification scheme does not encompass all T4SSs, but suffices here as we explore the evolutionary diversification of T4SSs. ...
... The T4BSS transporters evolved from I-type conjugation systems. The representative L. pneumophila Dot/Icm system, so named because the Isberg and Shuman groups independently discovered it and respectively named it Dot and Icm (defective in organelle trafficking/intracellular multiplication system), requires over >25 proteins of which only a few are VirB homologs (Marra et al. 1992;Sadosky et al. 1993;Berger and Isberg 1993;Brand et al. 1994;Nagai and Kubori 2011;Voth et al. 2012). The T4ASS and T4BSS classification scheme does not encompass all T4SSs, but suffices here as we explore the evolutionary diversification of T4SSs. ...
The bacterial type IV secretion systems (T4SSs) are a highly functionally and structurally diverse superfamily of secretion systems found in many species of Gram-negative and -positive bacteria. Collectively, the T4SSs can translocate DNA and monomeric and multimeric protein substrates to a variety of bacterial and eukaryotic cell types. Detailed phylogenomics analyses have established that the T4SSs evolved from ancient conjugation machines whose original functions were to disseminate mobile DNA elements within and between bacterial species. How members of the T4SS superfamily evolved to recognize and translocate specific substrate repertoires to prokaryotic or eukaryotic target cells is a fascinating question from evolutionary, biological, and structural perspectives. In this chapter, we will summarize recent findings that have shaped our current view of the biological diversity of the T4SSs. We focus mainly on two subtypes, designated as the types IVA (T4ASS) and IVB (T4BSS) systems that respectively are represented by the paradigmatic Agrobacterium tumefaciens VirB/VirD4 and Legionella pneumophila Dot/Icm T4SSs. We present current information about the composition and architectures of these representative systems. We also describe how these and a few related T4ASS and T4BSS members evolved as specialized nanomachines through acquisition of novel domains or subunits, a process that ultimately generated extensive genetic and structural mosaicism among this secretion superfamily. Finally, we present new phylogenomics information establishing that the T4BSSs are much more broadly distributed than initially envisioned.
... and Helicobacter pylori ( Fig. 2A). L. pneumophila, a cause of acute pneumonia in humans, utilizes a T4SS (termed Dot/Icm) to build an intracellular niche permissive for replication by delivering over 300 effector proteins with the capacity to modulate a myriad of host cell functions (Berger and Isberg, 1993;Brand et al., 1994;Hubber and Roy, 2010). Similarly, Coxiella burnetti, the cause of Q fever in humans, utilizes a related T4SS to survive and replicate within host cells (Kohler and Roy, 2015). ...
Many bacteria have evolved specialized nanomachines with the remarkable ability to inject multiple bacterially encoded effector proteins into eukaryotic or prokaryotic cells. Known as type III, type IV, and type VI secretion systems, these machines play a central role in the pathogenic or symbiotic interactions between multiple bacteria and their eukaryotic hosts, or in the establishment of bacterial communities in a diversity of environments. Here we focus on recent progress elucidating the structure and assembly pathways of these machines. As many of the interactions shaped by these machines are of medical importance, they provide an opportunity to develop novel therapeutic approaches to combat important human diseases.
... Legionella utilizes a Type 4 secretion (T4S) system termed "dot/ icm" [6][7][8] to translocate these effector proteins into the host cell 9,10 . T4S systems are comprised of protein complexes that assemble into a machine that traverses the bacterial inner membrane and cell envelope. ...
Legionella pneumophila, a causative agent of pneumonia, utilizes the Type 4B secretion (T4BS) system to translocate over 300 effectors into the host cell during infection. T4BS systems are encoded by a large gene cluster termed dot/icm, three components of which, DotL, DotM, and DotN, form the "coupling complex", which serves as a platform for recruitment of effector proteins. One class of effectors includes proteins containing Glu-rich/E-block sequences at their C terminus. However, the protein or region of the coupling complex mediating recruitment of such effectors is unknown. Here we present the crystal structure of DotM. This all alpha-helical structure exhibits patches of positively charged residues. We show that these regions form binding sites for acidic Glu-rich peptides and that mutants targeting these patches are defective in vivo in the translocation of acidic Glu-rich motif-containing effectors. We conclude that DotM forms the interacting surface for recruitment of acidic Glu-rich motif-containing Legionella effectors.
... In both amoebas and human alveolar macrophages, L. pneumophilia replicates in a parasitophorous vacuole termed the Legionella-containing vacuole (LCV; Swanson and Isberg, 1995; Figure 2). Biogenesis of the LCV requires translocation of Dot/Icm T4SS effector proteins (Berger and Isberg, 1993;Brand et al., 1994). While the precise details of how L. pneumophila evades endocytic maturation are unknown, the type IV effector protein VipD appears to play FIGURE 4 | Salmonella SPI-2 effector proteins participate in SCV formation. ...
Intracellular bacteria have developed numerous strategies to hijack host vesicular trafficking pathways to form their unique replicative niches. To promote intracellular replication, the bacteria must interact with host organelles and modulate host signaling pathways to acquire nutrients and membrane for the growing parasitophorous vacuole all while suppressing activation of the immune response. To facilitate host cell subversion, bacterial pathogens use specialized secretion systems to deliver bacterial virulence factors, termed effectors, into the host cell that mimic, agonize, and/or antagonize the function of host proteins. In this review we will discuss how bacterial effector proteins from Coxiella burnetii, Brucella abortus, Salmonella enterica serovar Typhimurium, Legionella pneumophila, Chlamydia trachomatis, and Orientia tsutsugamushi manipulate the endocytic and secretory pathways. Understanding how bacterial effector proteins manipulate host processes not only gives us keen insight into bacterial pathogenesis, but also enhances our understanding of how eukaryotic membrane trafficking is regulated.
... Isolates that were positive by FA in the polyvalent L. pneumophila conjugate were then further examined by FA with type-specific conjugates to L. pneumophila serogroups 1 to 6. If the FA results indicated a Legionella species, the isolate was examined for the cellular fatty acids characteristic of the Legionella species to confirm the identification, If the fatty acid profile demonstrated a pattern of a high percentage of branched-chain cellular fatty acids, and if the isolate did not grow on blood agar or produce browning on FG or yeast extract tyrosine agar, was gram negative, and was otherwise similar to Legionella, the isolate was considered to be an LLO 27,28 . ...
... Lp1 has been studied for the presence of a variety of virulence genes, such as intracellular multiplication/defective in organelle trafficking (icm/dot) (15,16), repeats in structural toxin (rtxA) (17), and Legionella vir homologue (lvh) (18). The icm/dot genes encode the type IV secretion system and are responsible for intracellular multiplication. ...
Importance:
In this study the dispersion, clonality and virulence of environmental isolates of Legionella pneumophila (L.pneumophila) serogroup (sg) 2-14 (Lp2-14) in Greece are investigated. Genetic variability of Lp 2-14 in the Greek environment was identified together with the presence of the pathogenicity loci at a high percentage of the isolates. Despite the high prevalence of Lp 2-14 in the Greek environment no clinical cases were reported may be due to underdiagnosis of the disease. Almost all the legionellosis cases are diagnosed in Greece by using the urine antigen test which is specific for Lp 1. It is an urgent need to improve the clinical diagnosis of legionellosis by introducing an effective diagnostic test for Lp2-14 in urine and by promoting the PCR examination of respiratory specimens from patients with compatible clinical symptoms.
... The nomenclature of the Dot/Icm T4SS is from the icm locus and the dot locus. The icm genetic locus first comprises four putative genes, the icmWXYZ, are important for Legionella intracellular multiplication [107,108]. In contrast, genetic locus which was designated to be dot, could complement the mutant with defective intracellular replication and an inability to establish the ER-derived LCV [109]. ...
Legionella is the causative agent of legionellosis or Legionnaires’disease. Infection with Legionella spp. is an important cause of community and hospital-acquired pneumonia. Infection with Legionella is also one of the most common causes of severe pneumonia in community settings, and Legionella is isolated in 40% of hospital-acquired pneumonia cases. Intense research on Legionella pneumophila (L. pneumophila) and other Legionella spp. over the last two decades provided insights into the evolution, ecology, epidemiology, and pathogenesis of this opportunistic pathogenic and facultative intracellular bacterium. An in-depth understanding of the ecology and virulence factors of Legionella spp. will contribute to an in-depth comprehension of its pathogenesis and efficient, sustainable elimination of the bacteria from anthropogenic water systems and might lead to the development of novel therapeutics. This review summarizes available information regarding the cellular microbiology, pathogenesis and virulence factors of Legionella spp. with an emphasis on the potential virulence factors and the contribution of these factors to the pathogenesis.
Legionella pneumophila is a gram‐negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase‐like metabolic changes via alternative sigma factors, and two‐component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.
The host type I interferon (IFN) response protects against a Legionella pneumophilia infection. Other pathogens inhibit type I IFN-mediated cell signaling; however, the interaction between this signaling pathway and L. pneumophila has not been described. This study demonstrates that L. pneumophila inhibits the IFN-β signaling pathway in an IFN-β dose-dependent manner. However, L. pneumophila does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-β to L. pneumophila-infected macrophages limited bacterial growth in a nitric oxide (NO)-dependent pathway since inhibiting NO production via a chemical inhibitor increased bacterial growth. Finally, this study demonstrates that the type IV secretion system of L. pneumophila is required to inhibit IFN-β-mediated cell signaling. In conclusion, this study describes a novel host cell signaling pathway inhibited by L. pneumophila, which improves the survival of bacteria in infected macrophages.
To prevail in the interaction with eukaryotic hosts, many bacterial pathogens use protein secretion systems to release virulence factors at the host–pathogen interface and/or deliver them directly into host cells. An outstanding example of the complexity and sophistication of secretion systems and the diversity of their protein substrates, effectors, is the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) Type IVB secretion system (T4BSS) of Legionella pneumophila and related species. Legionella species are facultative intracellular pathogens of environmental protozoa and opportunistic human respiratory pathogens. The Dot/Icm T4BSS translocates an exceptionally large number of effectors, more than 300 per L. pneumophila strain, and is essential for evasion of phagolysosomal degradation and exploitation of protozoa and human macrophages as replicative niches. Recent technological advancements in the imaging of large protein complexes have provided new insight into the architecture of the T4BSS and allowed us to propose models for the transport mechanism. At the same time, significant progress has been made in assigning functions to about a third of L. pneumophila effectors, discovering unprecedented new enzymatic activities and concepts of host subversion. In this review, we describe the current knowledge of the workings of the Dot/Icm T4BSS machinery and provide an overview of the activities and functions of the to-date characterized effectors in the interaction of L. pneumophila with host cells.
Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage co-culture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii , notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines.
Importance
Multi-drug-resistant pathogens are an emerging threat to human health. Since conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of “smarter” therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogen’s Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii , our study represents the first step in our pursuit towards precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.
Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics.
Bacteria of the genus Legionella are natural pathogens of amoebae that can cause a severe pneumonia in humans called Legionnaires’ Disease. Human disease results from inhalation of Legionella-contaminated aerosols and subsequent bacterial replication within alveolar macrophages. Legionella pathogenicity in humans has resulted from extensive co-evolution with diverse genera of amoebae. To replicate intracellularly, Legionella generates a replication-permissive compartment called the Legionella-containing vacuole (LCV) through the concerted action of hundreds of Dot/Icm-translocated effector proteins. In this review, we present a collective overview of Legionella pathogenicity including infection mechanisms, secretion systems, and translocated effector function. We also discuss innate and adaptive immune responses to L. pneumophila, the implications of Legionella genome diversity and future avenues for the field.
This is a book chapter published in 2006 as part of the Proceedings of the International Legionella Conference entitled: "Legionella: State of the Art 30 years after its recognition". The chapter describes ongoing research at the time, showing experimental evidence that supports the notion that the Hsp60 chaperonin of Legionella (named HtpB), is transported to the cell surface by a mechanism that involves passage through the periplasm and ultimately depends on the type IV secretion system Dot/Icm. In addition, using three different functional models of experimentation, it is shown that HtpB is capable to alter eukaryotic cell signalling pathways, actin polymerization/reorganization and mitochondrial trafficking. Several of these findings have been published in subsequent years (Narallah et al. 201, J. Bacteriol. 193: 4346-4360; Garduno et al. 2011, Front. Microbiol. 2:122. doi:10.3389/fmicb.2011.00122; Chong et al. 2009, Infect. Immun. 77: 4724-4739; and the book chapter: Valenzuela-Valderas, et al. 2016. Chapter 6: Legionella pneumophila chaperonin 60, an extra- and intra-cellular moonlighting virulence-related factor. In: Moonlighting Proteins: Novel Virulence Factors in Bacterial Infections [edited by B. Henderson]. John Wiley & Sons, Inc., Hoboken, NJ, pp. 111-134).
Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII heme-binding protein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.
During infection, the Legionnaires’ disease bacterium, Legionella pneumophila , survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism’s ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA . By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila -containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.
Within the human host, Legionella pneumophila replicates within alveolar macrophages, leading to pneumonia. However, L. pneumophila is an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles of L. pneumophila within the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome of L. pneumophila , which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors among Legionella species and >320 effector proteins in L. pneumophila into host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation of L. pneumophila to various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrict L. pneumophila , utilization of such hosts can also be instrumental in deciphering the mechanisms of failure of L. pneumophila to overcome restriction of certain protist hosts. Here, we review the interaction of L. pneumophila with its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge of L. pneumophila -protist host interaction and L. pneumophila ’s evolution to become a human pathogen.
The evolutionarily conserved processes of endosome-lysosome maturation and macroautophagy are established mechanisms that limit survival of intracellular bacteria. Similarly, another emerging mechanism is LC3-associated phagocytosis (LAP). Here we report that an intracellular vacuolar pathogen, Legionella dumoffii, is specifically targeted by LAP over classical endocytic maturation and macroautophagy pathways. Upon infection, the majority of L. dumoffii resides in ER-like vacuoles and replicate within this niche, which involves inhibition of classical endosomal maturation. The establishment of the replicative niche requires the bacterial Dot/Icm type IV secretion system (T4SS). Intriguingly, the remaining subset of L. dumoffii transiently acquires LC3 to L. dumoffii-containing vacuoles in a Dot/Icm T4SS-dependent manner. The LC3-decorated vacuoles are bound by an apparently undamaged single membrane, and fail to associate with the molecules implicated in selective autophagy, such as ubiquitin or adaptors. The process requires toll-like receptor 2, Rubicon, diacylglycerol signaling and downstream NADPH oxidases, whereas ULK1 kinase is dispensable. Together, we have discovered an intracellular pathogen, the survival of which in infected cells is limited predominantly by LAP. The results suggest that L. dumoffii is a valuable model organism for examining the mechanistic details of LAP, particularly induced by bacterial infection.
Drug-resistant bacterial pathogens represent an ever-increasing public health problem and call for the identification of novel anti-infective compounds. Environmental amoebae share many similarities with mammalian immune system phagocytes and therefore represent versatile and robust cellular models for screening and characterizing novel antibiotic, anti-virulence, or immune-boosting compounds. Using the amoeba Acanthamoeba castellanii, we developed robust fluorescence-based growth assays for the vacuolar pathogens Legionella pneumophila and Mycobacterium marinum. In screens for anti-virulence compounds, we discovered a hitherto unrealized antibiotic property for the β-lactone palmostatin M. Taken together, our studies revealed the utility of amoebae-based drug screening systems for the identification of novel anti-infective compounds.
The molecular analysis of Legionella pneumophila had been limited by the lack of methods to genetically manipulate the organism. Since bacteriophages and transduction have not been reported in L. pneumophila, methods of introducing DNA into the organism were limited to conjugation until 1990. Since then electroporation has rapidly replaced conjugation as the preferred method of introducing DNA into L. pneumophila for mutagenesis — mediated by transposons (Pope et al, 1994, Wiater et al, 1994, Pruckler et al, 1995, McClain and Engleberg, 1996, Hickey and Cianciotto, 1997) or by allelic exchange (Cianciotto and Fields, 1992, Sadosky et al, 1993, Abu Kwaik et al, 1997) and for complementation studies (Marra and Shuman, 1992, Berger et al, 1994, Abu Kwaik and Pederson, 1996).
Intracellular symbiosis is a wide-spread and important biological phenomenon in terms of genetic novelty, bringing about genetic changes that may be greater in magnitude than those which may result from mutation, hybridization or ploidy changes [Margulis, 1993; Sato et al., 1994], because symbiosis draws genomes from the entire biosphere [Jeon & Danielii, 1971; Gray, 1992-3; Maynard Smith & Szathmary, 1995]. Bacterial infection and intracellular survival inside eukaryotic cells have been extensively studied, but much remains still unknown about the mechanisms underlying the phenomena [Galan, 1994; Bull & Fogarty, 1995; Jeon, 1995-6; Russell, 1995; Finlay & Cossart, 1997].
Past outbreaks of Legionnaires' Disease and Pontiac Fever, i.e., legionellosis, in machining facilities have prompted intensive investigations to ascertain the source(s) of the etiologic agent - Legionella pneumophila. The outcomes of these investigations have been largely inconclusive, although cooling towers and hot water sources have been implicated as probable reservoirs of L. pneumophila. Metalworking fluid systems have been considered as possible sources of Legionella exposures. However, available data indicate that problematic Legionella species do not survive in metalworking fluids long enough to pose a health threat to machinists. Results from one laboratory study showed that L. pneumophila cultures that were inoculated into recirculating metalworking fluids did not proliferate but, instead, experienced decreases in viable L. pneumophila in 24-48 hrs or less. Low incidences of Legionnaires' Disease in susceptible workers are not consistent with what is known about risk factors. Ongoing research seeks to better understand water-dependent factors that lead to the proliferation and dissemination of Legionella bacteria in machining environments. This review examines potential sources of Legionella exposures in machining facilities.
Many bacterial pathogens use dedicated translocation systems to deliver arsenals of effector proteins to their hosts. Once inside the host cytosol, these effectors modulate eukaryotic cell biology to acquire nutrients, block microbial degradation, subvert host defenses, and enable pathogen transmission to other hosts. Among all bacterial pathogens studied to date, the gram-negative pathogen, Legionella pneumophila, maintains the largest arsenal of effectors, with over 330 effector proteins translocated by the Dot/Icm type IVB translocation system. In this review, I will discuss some of the recent work on understanding the consequences of this large arsenal. I will also present several models that seek to explain how L. pneumophila has acquired and subsequently maintained so many more effectors than its peers.
This chapter focuses on the various biochemical, immunological, and genetic strategies that are successfully employed by microbiologists to identify the bacterial pathogenicity factors. In addition, speculation on the utilization of several postgenomic strategies that are currently being developed is presented in an attempt to illuminate future methodologies, which should prove useful in further discoveries. A listing of each technique along with a brief description of some of their advantages and disadvantages is addresses in the chapter. The chapter also deals with the use of a broader definition of pathogenicity factor that includes any factor produced by a pathogen that plays a role in its survival, multiplication, or spread to new hosts. It also describes many of the genetic strategies that have been employed throughout the last few decades of the twentieth century to facilitate identification of bacterial pathogenicity factors. Each technique, as described, can result in identification of a bacterial gene that is required for production of a putative pathogenicity factor.
Infektionskrankheiten wie Diarrhö, Typhus, Keuchhusten, Tuberkulose, Lepra, Gastroenteritis, Meningitis, Lungenentzündung etc., die durch pathogene Bakterien verursacht werden, stellen weltweit eines der größten Gesundheitsprobleme für den Menschen dar. Trotz verfügbarer Therapeutika, insbesondere Antibiotika, sind diese Krankheiten nach wie vor die häufigste Ursache für Mortalität und Morbidität bei Menschen, v. a. in Ländern der III. Welt. Wie die jährlichen WHO-Berichte belegen, gehören in diesen Ländern bakterielle Darminfektionen bei Kleinkindern nach wie vor zu den wichtigsten Gründen für die hohe Kindersterblichkeit. Das Problem wird wesentlich verschärft durch die erschreckend hohe Zunahme an Resistenzen gegenüber den wichtigsten Antibiotika in vielen pathogenen Bakterien [Jacoby 1996].
Legionella pneumophila ist der Erreger der Legionärskrankheit. Die Pathogenität des Bakteriums basiert auf seiner Fähigkeit innerhalb menschlicher Lungenzellen zu überleben und sich zu vermehren. Demzufolge ist L. pneumophila nicht nur interessant als wichtiges Pathogen, sondern kann auch als Sonde verwendet werden, um allgemeine intrazelluläre Ereignisse zu untersuchen. Ein Beispiel hierfür ist die, durch das Pathogen gestörte, intrazelluläre Kommunikation zwischen den Organellen des endoplasmatischen Retikulums (ER) und dem Golgi Apparat (GA). In der vorliegenden Studie schlagen wir ein neues Modell vor, wie das Bakterium erfolgreich seine replikative Nische, die Legionella Vakuole (LV), innerhalb des Zytosols aufbauen könnte, um seine Ausbreitung zu garantieren. Um die Mechanismen für die erfolgreiche Ausbeutung der Wirtszelle gezielt untersuchen zu können, haben wir mit Hilfe von siRNA spezifisch verschiedene Wirtszellproteinen herunterreguliert und den Einfuß der Abwesenheit dieser Proteine auf die Vermehrung von L. pneumophila gemessen. Die Ergebnisse wiesen darauf hin, dass die LV möglicherweise den Golgi Apparat imitiert und auf diese Weise den zellulären Vesikeltransport umleitet. Diese Theorie wurde durch in silico Ergebnisse unterstützt, die in der Proteinsequenz des Legionella Effektor-Proteins LidA, das auf der Vakuole lokalisiert ist, ein SNARE-ähnliches Motiv zeigte. Dies weist auf ein auf der Vakuole lokalisiertes SNARE-Erkennungsmotiv hin, das notwendig sein könnte, um zelluläre Transportvesikel zu koppeln. Aus dem Wissen heraus, dass L. pneumophila in der Lage ist, die Aktivierung der zellulären Proteine Arf1 und Rab1 durch Phosphorylierung und Dephosphorylierung zu regulieren, machten wir uns auf die Suche nach Proteinen, die auf Infektion hin modifiziert werden. Die Kommunikation von Wirt und Pathogen über Phosphorylierung ist bekannt im Bezug auf pathogenspezifische Modifikation des Zytoskeletts und Signalkaskaden in der Anti-Apoptose. Für diese Studie wurde ein Antikörper verwendet, der spezifisch phosphorylierte Tyrosinreste erkennt. Dies resultierte in der Detektion einer Serin-Threonin-Kinase in der Amöbe Acanthamöba castellanii, die an einem Tyrosinrest phosphoryliert ist. Diese Amöben-Kinase wies in silico Homologie zu der humanen GS-Kinase 3 des Wnt-Signalwegs, bekannt aus der Forschung der embronalen Entwicklung bei Drosophila, auf. Der letzte Teil dieser Arbeit konzentrierte sich auf die, durch eine L. pneumophila-Infektion ausgelöste, anti-apoptotische Signalkaskade. Es ist bekannt, dass auf eine Infektion hin NF-kappaB aktiviert wird. Dies führt dazu, dass p65 in den Zellkern wandert und dort als Transkriptionsfaktor aktiv wird. Diese Translokation geschieht in 2 zeitversetzten Phasen. Eine Aktivierungsspitze wird nach dem Kontakt mir bakteriellem Flagellin gemessen, gefolgt, von einer dauerhaften Aktivierung, abhängig von einem funktionierenden Dot/ Icm Typ-IV-Translokationssystem. In dieser Arbeit stießen wir auf eine L. pneumophila Mutante, die den Dot/ Icm-Effektor SdbA nicht bildet, und die daraufhin NF-appaB nicht aktivieren kann. Diese Mutante war ebenfalls nicht in der Lage, sich in Epithelzellen zu vermehren. Dies ist außergewöhnlich, da das L. pneumophila Effektor Repertoire so redundant ist, dass die Abwesenheit eines einzigen Effektors selten einen so starken Einfluss auf die Replikation hat. All diese Ergebnisse zeigen zusammengenommen, auf wie vielen verschiedenen Ebenen L. pneumophila in der Lage ist, seine Wirtszelle zu manipulieren, um einerseits die nötige Nische für seine Vermehrung zu etablieren und andererseits die Zelle am Selbstmord zu hindern. Dies geschieht durch Imitation zellulärer Prozesse.
A lambda Insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(−), contained within the vector, can be excised by 11 or M13 helper phage. The excision process
elimlnates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This
is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the 11 bacteriophage
origin of replication (1). Six of 21 restriction sites In the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame,
can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated
and analyzed.
The lambda ZAP vector was tested by the preparation of a chicken liver cDNA Hbrary and the isolatlon of actin clones by screening
with oligonucleotide probes. Putative actin clones were excised from the lambda vector and Identified by DNA sequencing. The
ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion
proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.
A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.
The legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular parasite. Its interaction with phagocytes has characteristics in common with several other intracellular parasites. Critical aspects of L. pneumophila intracellular multiplication are evasion of lysosomal host cell defenses and the presence of a nutritionally appropriate environment. Following phagocytosis, wild-type L. pneumophila multiply within a specialized phagosome which does not fuse with secondary lysosomes. Mutants which have lost the ability to grow within phagocytes no longer cause disease in animals, indicating that the capacity to multiply intracellularly is important for pathogenesis. One such mutant, 25D, has been shown to be defective in inhibiting phagosome-lysosome fusion. This phagolysosomal environment is not conducive to Legionella growth. We report the isolation of a region of the L. pneumophila genome (icm, intracellular multiplication) which restores the capacity of 25D to multiply in human macrophages. The complemented mutants also regain the capacity to interfere with phagosome-lysosome fusion and to cause lethal pneumonia in guinea pigs.
Legionella pneumophila is a facultative intracellular bacterial pathogen that parasitizes human monocytes and alveolar macrophages. Previous studies from this laboratory have shown that monocyte complement receptors CR1 and CR3 and complement component C3 in serum mediate L. pneumophila phagocytosis. In this study, we have explored C3 fixation to L. pneumophila. We developed a whole-cell enzyme-linked immunosorbent assay (ELISA) to measure C3 fixation to the bacterial surface. By this assay, C3 fixes to L. pneumophila that are opsonized in fresh nonimmune serum, and C3 fixation takes place via the alternative pathway of complement activation. Immunoblot analysis of opsonized L. pneumophila indicated that C3 fixes selectively to specific acceptor molecules of L. pneumophila. Consistent with this, when nitrocellulose blots of whole L. pneumophila or bacterial components are incubated in fresh nonimmune serum, C3 fixes exclusively to the major outer membrane protein (MOMP) of L. pneumophila, a porin; C3 does not fix to L. pneumophila LPS on these blots. To further explore the role of MOMP in C3 fixation and phagocytosis, we reconstituted purified MOMP into liposomes. By the ELISA, MOMP-liposomes, but not plain liposomes lacking MOMP, avidly fix C3. Consistent with a dominant role for MOMP in C3 fixation, MOMP-liposomes form a C3 complex of the same apparent molecular weight as whole L. pneumophila in nonimmune serum. Opsonized radioiodinated MOMP-liposomes avidly adhere to monocytes, and adherence is dose dependent upon serum. By electron microscopy, opsonized MOMP-liposomes are efficiently phagocytized by human monocytes, and phagocytosis takes place by a conventional appearing form of phagocytosis. This study demonstrates that C3 fixes selectively to the MOMP of L. pneumophila, and that, in the presence of nonimmune serum, MOMP can mediate phagocytosis of liposomes and, potentially, phagocytosis of intact L. pneumophila by human monocytes.
We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.
Many strains of E.coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes. Such restriction can severely
inhibit the efficiency of cloning genomic DNAs. We have quantitatively evaluated a total of 39 E.coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems. Quantitative estimations of relative
tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical
recombinant cloning situations. Host strains are recommended for different recombinant cloning requirements. These data also
provide a rational basis for future construction of ‘ideal’ hosts combining optimal methylation tolerance with additional
advantageous mutations.
To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.
A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(-), contained within the vector, can be excised by f1 or M13 helper phage. The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the f1 bacteriophage origin of replication (1). Six of 21 restriction sites in the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.
Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.
The DNA sequence of 168 promoter regions (−;50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter
had been defined by genetic (promoter mutations) or biochemical (5′ end determination) criteria. A consensus promoter sequence
based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations.
In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following
general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.
We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KB k2 , the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted
to within a factor of ±4.1 in KB k2 over a range of 104 in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating
possible promoters in DNA sequence files.
Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.
The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
This compendium of techniques coverage of state-of-the-art developments in molecular biology, with over 600 pages of information contributed by a wide range of authorities.
Charcoal-yeast extract agar is a new bacteriological medium that supports excellent growth of the Legionella pneumophila. It results from modifications made in an existing L. pneumophila medium, F-G agar. Yeast extract, instead of an acid hydrolysate of casein, serves as the protein source. Beef extractives and starch are not added. Activated charcoal (Norit A or Norit SG) is included at 0.20% (wt/vol). Comparison of charcoal-yeast extract and F-G agars showed that a greater number of colony-forming units of L. pneumophila was recovered from a standardized tissue inoculum on charcoal-yeast extract agar (4.35 x 10(6) colony-forming units) than on F-G agar (4.85 x 10(4) colony-forming units). Macroscopic colonies of L. pneumophila were visible on the new medium within 3 days, whereas 4 days of growth was required on F-G agar.
The major outer membrane protein of Legionella pneumophila is composed of 28- and 31-kDa subunits cross-linked by interchain disulfide bonds. The oligomer is covalently anchored to the underlying peptidoglycan via the 31-kDa subunit. We have cloned the structural gene ompS encoding both proteins. Oligonucleotide probes synthesized from the codons of the N-terminal amino acid sequence of purified 28- and 31-kDa subunits were used to identify cloned sequences. A 2.9-kb HindIII fragment cloned into pBluescript (clone H151) contained the ompS gene. Nucleotide sequence analysis revealed an open reading frame of 891 bp encoding a polypeptide of 297 amino acids. A leader sequence of 21 amino acids was identified, and the mature protein contained 276 amino acids. The deduced amino acid sequence of OmpS matched the experimentally determined amino acid sequence (32 amino acids), with the exception of two cysteine residues. The deduced amino acid sequence was rich in glycine and aromatic amino acids and contained four cysteine residues, two in the amino terminus and two in the carboxy region. Primer extension analysis (total RNA from L. pneumophila) identified the transcription start at 96 to 98 bp upstream of the translation start, but no Escherichia coli-like promoter sequences were evident. While an mRNA transcript from clone H151 was detected, no cross-reactive protein was detected by immunoblotting with either monoclonal or polyclonal antibody. Attempts to subclone the gene in the absence of the putative promoter region (i.e., under the control of the lac promoter) proved unsuccessful, possibly because of overproduction lethality in E. coli. The ompS DNA sequence was highly conserved among the serogroups of L. pneumophila, and related species also exhibited homology in Southern blot analysis at a moderately high stringency. Evidence is presented to suggest that this gene may be environmentally regulated in L. pneumophila.
A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.
The facultative intracellular pathogen, Legionella pneumophila, multiplies within and kills human monocytes and alveolar macrophages. We show that L. pneumophila strain Philadelphia-1 infects, multiplies within and kills the promyelocyte HL-60 cell line after its differentiation into macrophage-like cells. The characteristics of the interaction between L. pneumophila and differentiated HL-60 cells closely resemble those between L. pneumophila and human peripheral blood monocytes. With both cell types, C receptors and serum C mediate attachment of L. pneumophila, which are taken up by coiling phagocytosis. The replicative phagosome is lined with ribosomes; intracellular multiplication is iron-dependent; and replicating bacteria ultimately destroy the host cell. As in human monocytes, an avirulent mutant derivative of L. pneumophila Philadelphia-1, 25D, does not replicate in and is not cytopathic for differentiated HL-60 cells. Differentiated HL-60 cells therefore provide a convenient and faithful model for the study of L. pneumophila-mononuclear phagocyte interaction.
Infection by Legionella pneumophila is believed to depend upon its ability to multiply within host alveolar macrophages. To investigate this, a site-specific
mutation was introduced into a gene (mip) that encodes a 24,OOO-Da surface protein; an 80-fold loss of infectivity for both U937 cells and explanted human alveolar
macrophages was observed. Further phenotypic analysis of the mutant strain has failed to show alterations in bacterial factors
(e.g., proteinase, lipopolysaccharide) that have suspected roles in virulence. To substantiate that this mutation also results
in reduced virulence in animals, the lethality and clinical illnesses produced by the parent and mutant L. pneumophila strains were compared in guinea pigs after intratracheal inoculation. The mutant strain produced fewer illnesses, slower-progressing
disease, and fewer lethal infections than either the parent strain or a derivative of the mutant strain with the wild-type
mip gene reintroduced. When sublethal inocula of the three strains were used, the mutant bacteria were recovered in slightly
lower numbers from lung homogenates and in significantly lower numbers from the spleen, at 48 h, than were the other two test
strains. Thus mip seems to be necessary for full virulence of L. pneumophila and may represent the first genetically defined virulence factor in this species.
The REP sequence is a highly conserved inverted repeat that is present in about 25% of all E. coli transcription units. We show that the REP sequence can stabilize upstream RNA, independently of any other sequences, by protection from 3'-5' exonuclease attack. The REP sequence is frequently responsible for the differential stability of different segments of mRNA within an operon. We demonstrate that REP-stabilized mRNA can be translated in vivo and that cloning the REP sequence downstream of a gene can increase protein synthesis. This provides direct evidence that alterations in mRNA stability can play a role in determining bacterial gene expression. The implications of these findings for the mechanisms of mRNA degradation and for the role of RNA stability in the regulation of gene expression are discussed.
DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.
We established a model of the bacteria-macrophage interaction to study the cellular basis of Legionella pneumophila pathogenesis and to characterize avirulent L. pneumophila. We found that U937 cells, which are derived from a human histiocytic lymphoma cell line, support intracellular growth of L. pneumophila with a doubling time of 6 h, and that sustained intracellular growth is associated with a cytopathic effect (CPE) that can be detected by microscopic examination and quantified with the vital stain 3-(4,5-dimethyl thiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (MTT). An L. pneumophila isolate obtained directly from infected guinea-pig spleens can grow and produce CPE in these cells, destroying most of the cell layer after 72 h of growth. Only 10(6) organisms of this strain are required to kill 50% of guinea-pigs inoculated by the intraperitoneal route. In contrast, an avirulent isolate derived by 203 successive plate passages of the same strain can neither kill guinea-pigs at an intraperitoneal inoculum of 10(7) nor grow or produce CPE in U937 cells. Since the cells were able to differentiate between a virulent and an avirulent strain of L. pneumophila, we conclude that U937 cells are an appropriate model system for study of the bacteria-macrophage interaction.
A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.
The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
The specificity of the signal sequence cleavage reaction has been postulated to reside in a signal peptidase active site that can bind only to particular (i, i + 2) pairs of amino acids. In this paper, we present further patterns of non-random amino acid utilization in a region around in vivo cleavage sites, and show that they can be interpreted in terms of selection acting to reduce the number of potential competing sites in the vicinity of the correct one.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
The interactions between the L. pneumophila phagosome and monocyte lysosomes were investigated by prelabeling the lysosomes with thorium dioxide, an electron-opaque colloidal marker, and by acid phosphatase cytochemistry. Phagosomes containing live L. pneumophila did not fuse with secondary lysosomes at 1 h after entry into monocytes or at 4 or 8 h after entry by which time the ribosome-lined L. pneumophila replicative vacuole had formed. In contrast, the majority of phagosomes containing formalin-killed L. pneumophila, live Streptococcus pneumoniae, and live Escherichia coli had fused with secondary lysosomes by 1 h after entry into monocytes. Erythromycin, a potent inhibitor of bacterial protein synthesis, at a concentration that completely inhibits L. pneumophila intracellular multiplication, had no influence on fusion of L. pneumophila phagosomes with secondary lysosomes. However, coating live L. pneumophila with antibody or with antibody and complement partially overcame the inhibition of fusion. Also activating the monocytes promoted fusion of a small proportion of phagosomes containing live L. pneumophila with secondary lysosomes. Acid phosphatase cytochemistry revealed that phagosomes containing live L. pneumophila did not fuse with either primary or secondary lysosomes. In contrast to phagosomes containing live bacteria, the majority of phagosomes containing formalin-killed L. pneumophila were fused with lysosomes by acid phosphatase cytochemistry. The capacity of L. pneumophila to inhibit phagosome-lysosome fusion may be a critical mechanism by which the bacterium resists monocyte microbicidal effects.
Phagocytosis of Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in human mononuclear phagocytes and causes Legionnaires' disease, occurs by a novel mechanism. A phagocyte pseudopod coils around the bacterium as the organism is internalized. Human monocytes, alveolar macrophages, and polymorphonuclear leukocytes all phagocytize L. pneumophila by this unusual process, termed "coiling phagocytosis," and these leukocytes phagocytize not only live L. pneumophila in this way, but also formalin-killed, glutaraldehyde-killed, and heat-killed L. pneumophila. In contrast, under the same experimental conditions, monocytes phagocytize Streptococcus pneumoniae, encapsulated and unencapsulated E. coli, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Neisseria gonorrhoeae, and Neisseria meningitidis by conventional phagocytosis. Treatment of L. pneumophila with high-titer anti-L. pneumophila antibody abolishes coiling phagocytosis; such bacteria are internalized by conventional phagocytosis. These experiments raise the possibility that a surface component of L. pneumophila mediates the unusual response by the phagocyte. Such a component, if elaborated in vivo, might be responsible for extrapulmonary manifestations of Legionnaires' disease suspected of being toxin-mediated.
To define mechanisms by which polysaccharide capsules confer enhanced virulence on gram-negative bacteria, we examined the effect of the Escherichia coli capsule on complement fixation to the bacterial surface and on phagocytosis and killing of these bacteria by mouse macrophages and human polymorphonuclear leukocytes (PMN) and monocytes. When E. coli were attached to mouse macrophages with concanavalin A, the macrophages readily phagocytosed unencapsulated but not encapsulated bacteria even in the presence of fresh mouse serum; macrophages did not phagocytose encapsulated E. coli unless antibacterial or anti-Con A antibody was added. Similarly, when these bacteria were attached to human PMN with Con A, PMN ingested unencapsulated but not encapsulated E. coli. PMN phagocytosed and killed encapsulated serum-resistant E. coli only in the presence of both complement and antibacterial antibody; PMN phagocytosed and killed unencapsulated E. coli of the same strain in the presence of complement alone. Fluorescence microscopy showed that antibody had to be present for encapsulated but not unencapsulated E. coli to fix complement to its surface. To examine the role of the complement receptors of human PMN and monocytes in phagocytosis and killing of encapsulated E. coli, we used human and rabbit antibacterial immunoglobulin (Ig)M to fix complement to the bacteria. PMN and monocytes phagocytosed and killed encapsulated E. coli in the presence of both IgM and complement, but not in the presence of either serum opsonin alone. In the presence of antibacterial IgG, PMN and monocytes required complement to effectively phagocytose and kill the E. coli. We conclude that (a) attachment by itself results in ingestion of unencapsulated but not encapsulated E. coli; (b) under physiologic conditions, E. coli are not phagocytosed or killed the absence of antibody, the E. coli capsule blocks complement fixation to the bacterial surface probably by masking surface components, such as lipopolysaccharide, capable of activating the complement pathway; (d) the E. coli capsule imposes a requirement for specific antibacterial antibody for complement fixation; and (e) the complement receptor of human PMN and monocytes mediates phagocytoses of complement-coated encapsulated bacteria and is the primary mediator of phagocytosis and killing of these bacteria.
We have studied the interaction between virulent egg yolk-grown Legionella pneumophila Philadelphia 1 and human blood monocytes in vitro. The leukocytes were cultured in antibiotic-free tissue culture medium supplemented with 15% autologous human serum.L. pneumophila multiplied several logs, as measured by colony-forming units, when incubated with monocytes or mononuclear cells; the mid-log phase doubling time was 2 h. The level to which L. pneumophila multiplied was proportional to the number of mononuclear cells in the culture. L. pneumophila multiplied only in the adherent fraction of the mononuclear cell population indicating that monocytes but not lymphocytes support growth of the bacteria. Peak growth of L. pneumophila was correlated with destruction of the monocyte monolayer. By fluorescence microscopy using fluorescein conjugated rabbit anti-L. pneumophila antiserum, the number of monocytes containing L. pneumophila increased in parallel with bacterial growth in the culture. At the peak of infection, monocytes were packed full with organisms. By electron microscopy, L. pneumophila in such monocytes were found in membrane-bound cytoplasmic vacuoles studded with structures resembling host cell ribosomes. Several lines of evidence indicate that L. pneumophila grows within monocytes. (a) In the absence of leukocytes, L. pneumophila did not grow in tissue culture medium with or without serum even if the medium was conditioned by monocytes. (b) L. pneumophila did not grow in sonicated mononuclear cells. Lysis of these cells at various times during logarithmic growth of L. pneumophila was followed by cessation of bacterial multiplication. Growth resumed when intact mononuclear cells were added back to the culture. (3) In parabiotic chambers separated by 0.1-mum Nuclepore filters, L. pneumophila multiplied only when placed on the same side of the filter as mononuclear cells. These findings indicate that L. pneumophila falls into a select category of bacterial pathogens that evade host defenses by parasitizing monocytes. It remains to be determined whether cell-mediated immunity plays a dominant role in host defense against L. pneumophila as it does against other intracellular pathogens.
Legionella pneumophila dot mutations cause defects in intracellular targeting of the microorganism within cultured macrophages. Each of the previously characterized dot mutations was shown to be complemented by a single open reading frame designated dotA. The defects caused by the mutations appear to be due to disrupted function of the predicted 1048-amino-acid residue DotA protein, and not by polarity effects on a downstream gene. Complementation studies indicated that the product of the dotA53 mutation results in a partially functional DotA protein, consistent with a stable N-terminal fragment having biological activity.
Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome-lysosome fusion and association of host cell organelles with bacteria-containing phagosomes ('recruitment'). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome-lysosome fusion to mutants belonging to both phenotypic classes.
The HL-60 mode! for the interaction of human macrophages with the Legionnaires' disease bacterium Identification of a Legioneita pneumophita locus required for intracelluiar multiplication in human macro-phages
- A Marra
- M A Horwitz
- H A Shuman
- A Marra
- S J Blander
- M A Howitz
- H Shuman
Marra, A,, Horwitz, M.A., and Shuman. H.A. (1990) The HL-60 mode! for the interaction of human macrophages with the Legionnaires' disease bacterium. J Immunol 144: 2738-2744, Marra, A,, Blander, S.J,, Howitz, M.A,, and Shuman, H,A. (1992) Identification of a Legioneita pneumophita locus required for intracelluiar multiplication in human macro-phages, Proc NatI Acad Sci USA 89: 9607-9611.
Purification of Legioneiia pneumophila major outer mem-brane protein and demonstration that it is a porin Genetic and DNA sequence analysis of the kanamycin resistance transposon Jn903
- J Gabay
- E Blake
- M Niles
- D Horwitz
Gabay, J,E,, Blake, M,, Niles, W,D,, and Horwitz, M,A. (1985) Purification of Legioneiia pneumophila major outer mem-brane protein and demonstration that it is a porin. J Sacrer/o/162: 85-91, Grindley, N.D,F., and Joyce, CM, (1980) Genetic and DNA sequence analysis of the kanamycin resistance transposon Jn903. Proc NatI Acad Sci USA 77: 7176-7180.
A simple method for displaying the hydropathic character of a protein Molecuiar Cloning: A Laboratory Manual. Cold Spring Harbor
- J Kyte
- R Doolittle
- T Maniatis
- E F Fritsch
- J Sambrook
Kyte, J., and Doolittle, R.F, (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biot 157: 105-132, Maniatis, T., Fritsch, E.F,, and Sambrook, J. (1982) Molecuiar Cloning: A Laboratory Manual. Cold Spring Harbor. New York: Cofd Spring Harbor Laboratoiy Press.
Experiments in Molecular Genetics A series of wide-host-range low-copy-number vectors that allow direct screening tor recombinants
- J H Miiler
- S C Brand
- A B Sadosky
- K A Shuman Morales
- M Baeckmann
Miiler, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York: Cold Spring Harbor Laboratory Press. 808 S. C, Brand, A. B. Sadosky and K A. Shuman Morales, V,M,, Baeckmann, A,, and Bagdasarian, M, (1991) A series of wide-host-range low-copy-number vectors that allow direct screening tor recombinants. Gene 97: 39-47.