scholarly journals Legionella pneumophila CRISPR-Cas suggests recurrent encounters with one or more phages in the family Microviridae .

2021 ◽  
Author(s):  
Shayna R. Deecker ◽  
Malene L. Urbanus ◽  
Beth Nicholson ◽  
Alexander W. Ensminger
Keyword(s):  
Legionella Pneumophila ◽  
Sequence Similarity ◽  
Mobile Element ◽  
Current Data ◽  
Significant Sequence Similarity ◽  
Remediation Strategies ◽  
The Family ◽  
Legionnaires Disease ◽  
Outstanding Question

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires’ disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella Mobile Element-1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila ’s adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants - all targeted by previously and newly identified CRISPR-Cas spacers - but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae . These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires’ disease is an often-fatal pneumonia caused by Legionella pneumophila , which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the “missing” L. pneumophila phages belong to the microvirus subfamily Gokushovirinae . Gokushoviruses are known to infect another intracellular pathogen, Chlamydia . How do gokushoviruses access L. pneumophila (and Chlamydia ) inside their “cozy niches”? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

scholarly journals Legionella pneumophila CRISPR-Cas suggests recurrent encounters with Gokushovirinae

2021 ◽  
Author(s):  
Shayna R. Deecker ◽  
Malene L. Urbanus ◽  
Beth Nicholson ◽  
Alexander W. Ensminger
Keyword(s):  
Causative Agent ◽  
Legionella Pneumophila ◽  
Molecular Mechanisms ◽  
Human Lung ◽  
Mobile Element ◽  
Water Systems ◽  
Current Data ◽  
Remediation Strategies ◽  
Legionnaires Disease ◽  
Outstanding Question

ABSTRACTLegionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires’ disease. This pathogen and its ability to cause disease is closely tied to its environmental encounters. From phagocytic protists, L. pneumophila has “learned” how to avoid predation and exploit conserved eukaryotic processes to establish an intracellular replicative niche. Legionnaires’ disease is a product of these evolutionary pressures as L. pneumophila uses the same molecular mechanisms to replicate in grazing protists and in macrophages of the human lung. L. pneumophila growth within protists also provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems has been an episomal element that we previously named Legionella Mobile Element-1 (LME-1). In this study, we have identified over 150 CRISPR-Cas systems across 600 isolates, to establish the clearest picture yet of L. pneumophila’s adaptive defenses. By leveraging the sequence of 1,500 unique spacers, we can make two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1 and the heretofore “missing” L. pneumophila phages are most likely lytic gokushoviruses.IMPORTANCEThe causative agent of Legionnaires’ disease, an often-fatal pneumonia, is an intracellular bacterium, Legionella pneumophila, that normally grows inside amoebae and other freshwater protists. Unfortunately for us, this has two major consequences: the bacterium can take what it has learned in amoebae and use similar strategies to grow inside our lungs; and these amoebae can protect Legionella from various forms of chemical and physical disinfection regimes. Legionella are ubiquitous in the environment and frequently found in man-made water systems. Understanding the challenges to Legionella survival before it reaches the human lung is critical to preventing disease.We have leveraged our earlier discovery that L. pneumophila CRISPR-Cas systems are active and adaptive – meaning that they respond to contemporary threats encountered in the environment. In this way, CRISPR arrays can be considered genomic diaries of past encounters, with spacer sequences used to identify elements that may impinge on the pathogen’s survival. One outstanding question in the field is whether L. pneumophila is susceptible to phage, given the presumptive protection provided by intracellular replication within its eukaryotic hosts. In this work, we use CRISPR spacer sequences to suggest that the heretofore “missing” L. pneumophila phage are most likely lytic gokushoviruses. Such information is critical to the long-term goal of developing of new strategies for preventing colonization of our water systems by Legionella and subsequent human exposure to the pathogen.

An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12

1992 ◽  
Vol 12 (1) ◽  
pp. 56-67
Author(s):  
D A Maslov ◽  
N R Sturm ◽  
B M Niner ◽  
E S Gruszynski ◽  
M Peris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Sequence Similarity ◽  
Rich Region ◽  
Leishmania Tarentolae ◽  
Significant Sequence Similarity ◽  
The Family ◽  
Intergenic Regions ◽  
Ribosomal Protein S12

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.

scholarly journals Flagellum of Legionella pneumophilaPositively Affects the Early Phase of Infection of Eukaryotic Host Cells

2001 ◽  
Vol 69 (4) ◽  
pp. 2116-2122 ◽  
Author(s):  
Claudia Dietrich ◽  
Klaus Heuner ◽  
Bettina C. Brand ◽  
Jörg Hacker ◽  
Michael Steinert
Keyword(s):  
Electron Microscopy ◽  
Legionella Pneumophila ◽  
Kanamycin Resistance ◽  
Etiologic Agent ◽  
Polyclonal Antiserum ◽  
Host Cells ◽  
Kanamycin Resistance Cassette ◽  
Legionnaires Disease ◽  
Invasion Capacity

ABSTRACT Legionella pneumophila, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology ofLegionella, the flaA gene of L. pneumophila Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant L. pneumophila Corby KH3 was nonflagellated. The redelivery of the intact flaA gene into the chromosome (L. pneumophila Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the flaA mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of L. pneumophila positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.

scholarly journals Complete Genome Sequences of Six Legionella pneumophila Isolates from Two Collocated Outbreaks of Legionnaires’ Disease in 2005 and 2008 in Sarpsborg/Fredrikstad, Norway

2016 ◽  
Vol 4 (6) ◽  
Author(s):  
Marius Dybwad ◽  
Tone Aarskaug ◽  
Else-Marie Fykse ◽  
Elisabeth Henie Madslien ◽  
Janet Martha Blatny
Keyword(s):  
Comparative Genomics ◽  
Genetic Relationship ◽  
Legionella Pneumophila ◽  
Complete Genome ◽  
Sequence Similarity ◽  
Secretion Systems ◽  
Environmental Isolates ◽  
Genome Sequences ◽  
High Sequence Similarity ◽  
Legionnaires Disease

Here, we report the complete genome sequences of Legionella pneumophila isolates from two collocated outbreaks of Legionnaires’ disease in 2005 and 2008 in Sarpsborg/Fredrikstad, Norway. One clinical and two environmental isolates were sequenced from each outbreak. The genome of all six isolates consisted of a 3.36 Mb-chromosome, while the 2005 genomes featured an additional 68 kb-episome sharing high sequence similarity with the L. pneumophila Lens plasmid. All six genomes contained multiple mobile genetic elements including novel combinations of type-IVA secretion systems. A comparative genomics study will be launched to resolve the genetic relationship between the L. pneumophila isolates.

scholarly journals Legionella pneumophila Entry GenertxA Is Involved in Virulence

2001 ◽  
Vol 69 (1) ◽  
pp. 508-517 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Luiz E. Bermudez ◽  
Sahar H. El-Etr ◽  
Gerald E. Duhamel ◽  
Jeffrey D. Cirillo
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Integrin Receptors ◽  
Legionnaires Disease ◽  
Cell Spread ◽  
Gain Access

ABSTRACT Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1,enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2 integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry genertxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

scholarly journals Humidifier-associated paediatric Legionnaires' disease, Israel, February 2012

2012 ◽  
Vol 17 (41) ◽  
Author(s):  
J Moran-Gilad ◽  
T Lazarovitch ◽  
M Mentasti ◽  
T Harrison ◽  
M Weinberger ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Cold Water ◽  
Tap Water ◽  
Fatal Case ◽  
Control Measures ◽  
Free Standing ◽  
Legionnaires Disease

We report a fatal case of community-acquired Legionnaires' disease in an infant aged under six months. Epidemiological and microbiological investigations suggested that a free-standing cold water humidifier using domestic tap water contaminated with Legionella pneumophila serogroup 1 served as a vehicle for infection. These findings were corroborated by sequence-based typing (SBT). Humidifier-associated Legionnaires' disease can be prevented by appropriate control measures. This case also illustrates the emerging role of SBT in the investigation of legionellosis.

scholarly journals Legionnaires’ Disease Mortality in Guinea Pigs Involves the p45 Mobile Genomic Element

2019 ◽  
Vol 220 (10) ◽  
pp. 1700-1710
Author(s):  
Lanette M Christensen ◽  
Preeti Sule ◽  
Suat L G Cirillo ◽  
Madison Strain ◽  
Quinci Plumlee ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Guinea Pigs ◽  
Mobile Element ◽  
Interferon Γ ◽  
Secretion Systems ◽  
Proinflammatory Response ◽  
Disease Mortality ◽  
Genomic Element ◽  
Fatal Form ◽  
Legionnaires Disease

AbstractBackgroundLegionella can cause Legionnaires’ disease, a potentially fatal form of pneumonia that occurs as sporadic epidemics. Not all strains display the same propensity to cause disease in humans. Because Legionella pneumophila serogroup 1 is responsible for >85% of infections, the majority of studies have examined this serogroup, but there are 3 commonly used laboratory strains: L pneumophila serogroup 1 Philadelphia (Phil-1)-derived strains JR32 and Lp01 and 130b-derived strain AA100.MethodsWe evaluated the ability of Phil-1, JR32, Lp01, and AA100 to cause disease in guinea pigs.ResultsWe found that, although Phil-1, JR32, and AA100 cause an acute pneumonia and death by 4 days postinfection (100%), strain Lp01 does not cause mortality (0%). We also noted that Lp01 lacks a mobile element, designated p45, whose presence correlates with virulence. Transfer of p45 into Lp01 results in recovery of the ability of this strain to cause mortality, leads to more pronounced disease, and correlates with increased interferon-γ levels in the lungs and spleens before death.ConclusionsThese observations suggest a mechanism of Legionnaires’ disease pathogenesis due to the presence of type IVA secretion systems that cause higher mortality due to overinduction of a proinflammatory response in the host.

scholarly journals Legionella Contamination in Hot Water of Italian Hotels

2005 ◽  
Vol 71 (10) ◽  
pp. 5805-5813 ◽  
Author(s):  
Paola Borella ◽  
Maria Teresa Montagna ◽  
Serena Stampi ◽  
Giovanna Stancanelli ◽  
Vincenzo Romano-Spica ◽  
...  
Keyword(s):  
Risk Factors ◽  
Legionella Pneumophila ◽  
Water Systems ◽  
Hot Water ◽  
Cross Sectional ◽  
Multicenter Survey ◽  
Multivariate Logistic Model ◽  
Legionnaires Disease ◽  
Building Characteristics

ABSTRACT A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of ≥103 CFU liter−1, and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L. pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.

scholarly journals Immunomodulatory Role of Endogenous Interleukin-18 in Gamma Interferon-Mediated Resolution of Replicative Legionella pneumophila Lung Infection

2000 ◽  
Vol 68 (12) ◽  
pp. 6567-6573 ◽  
Author(s):  
Joan K. Brieland ◽  
Craig Jackson ◽  
Steve Hurst ◽  
David Loebenberg ◽  
Tony Muchamuel ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Lavage Fluid ◽  
Lung Infection ◽  
Gamma Interferon ◽  
Pcr Analysis ◽  
Interleukin 18 ◽  
Legionnaires Disease ◽  
Ifn Γ

ABSTRACT The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-γ)-mediated resolution of replicativeLegionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (106 L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-γ mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior toL. pneumophila infection inhibited induction of intrapulmonary IFN-γ production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-γ and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-γ in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.

scholarly journals An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12.

1992 ◽  
Vol 12 (1) ◽  
pp. 56-67 ◽  
Author(s):  
D A Maslov ◽  
N R Sturm ◽  
B M Niner ◽  
E S Gruszynski ◽  
M Peris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Sequence Similarity ◽  
Rich Region ◽  
Leishmania Tarentolae ◽  
Significant Sequence Similarity ◽  
The Family ◽  
Intergenic Regions ◽  
Ribosomal Protein S12

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.

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