scholarly journals Virulence Phenotypes of Legionella pneumophila Associated with Noncoding RNA lpr0035

2012 ◽  
Vol 80 (12) ◽  
pp. 4143-4153 ◽  
Author(s):  
Deepak Jayakumar ◽  
Julie V. Early ◽  
Howard M. Steinman
Keyword(s):  
Legionella Pneumophila ◽  
Noncoding Rna ◽  
Deletion Mutant ◽  
Acanthamoeba Castellanii ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Open Reading Frames ◽  
Causative Organism ◽  
Content Type ◽  
Intracellular Multiplication

ABSTRACTThe Philadelphia-1 strain ofLegionella pneumophila, the causative organism of Legionnaires' disease, contains a recently discovered noncoding RNA, lpr0035. lpr0035 straddles the 5′ chromosomal junction of a 45-kbp mobile genetic element, pLP45, which can exist as an episome or integrated in the bacterial chromosome. A 121-bp deletion was introduced in strain JR32, a Philadelphia-1 derivative. The deletion inactivated lpr0035, removed the 49-bp direct repeat at the 5′ junction of pLP45, and locked pLP45 in the chromosome. Intracellular multiplication of the deletion mutant was decreased by nearly 3 orders of magnitude inAcanthamoeba castellaniiamoebae and nearly 2 orders of magnitude in J774 mouse macrophages. Entry of the deletion mutant into amoebae and macrophages was decreased by >70%. The level of entry in both hosts was restored to that in strain JR32 by plasmid copies of two open reading frames immediately downstream of the 5′ junction and plasmid lpr0035 driven by its endogenous promoter. When induced from atacpromoter, plasmid lpr0035 completely reversed the intracellular multiplication defect in macrophages but was without effect in amoebae. These data are the first evidence of a role for noncoding RNA lpr0035, which has homologs in six otherLegionellagenomes, in entry ofL. pneumophilainto amoebae and macrophages and in host-specific intracellular multiplication. The data also demonstrate that deletion of a direct-repeat sequence restricts the mobility of pLP45 and is a means of studying the role of pLP45 mobility inLegionellavirulence phenotypes.

scholarly journals Environmental Mimics and the Lvh Type IVA Secretion System Contribute to Virulence-Related Phenotypes of Legionella pneumophila

2006 ◽  
Vol 75 (2) ◽  
pp. 723-735 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Shuqing Liu ◽  
Carolina B. Gabbai ◽  
Zeah Venitelli ◽  
Howard M. Steinman
Keyword(s):  
Stationary Phase ◽  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Resistant Bacteria ◽  
Causative Organism ◽  
Legionnaires Disease ◽  
Intracellular Multiplication ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

ABSTRACT Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.

scholarly journals Multiple Legionella pneumophila Type II Secretion Substrates, Including a Novel Protein, Contribute to Differential Infection of the Amoebae Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria lovaniensis

2013 ◽  
Vol 81 (5) ◽  
pp. 1399-1410 ◽  
Author(s):  
Jessica Y. Tyson ◽  
Meghan M. Pearce ◽  
Paloma Vargas ◽  
Sreya Bagchi ◽  
Brendan J. Mulhern ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Host Cells ◽  
Type Ii Secretion ◽  
Broad Host Range ◽  
Type Ii ◽  
Content Type ◽  
Intracellular Multiplication ◽  
Hartmannella Vermiformis ◽  
Naegleria Lovaniensis

ABSTRACTType II protein secretion (T2S) byLegionella pneumophilais required for intracellular infection of host cells, including macrophages and the amoebaeAcanthamoeba castellaniiandHartmannella vermiformis. Previous proteomic analysis revealed that T2S byL. pneumophila130b mediates the export of >25 proteins, including several that appeared to be novel. Following confirmation that they are unlike known proteins, T2S substrates NttA, NttB, and LegP were targeted for mutation.nttAmutants were impaired for intracellular multiplication inA. castellaniibut notH. vermiformisor macrophages, suggesting that novel exoproteins which are specific toLegionellaare especially important for infection. Because the importance of NttA was host cell dependent, we examined a panel of T2S substrate mutants that had not been tested before in more than one amoeba. As a result, RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all proved to be required for optimal intracellular multiplication inH. vermiformisbut notA. castellanii. Further examination of anlspFmutant lacking the T2S apparatus documented that T2S is also critical for infection of the amoebaNaegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA, were defective inN. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA inH. vermiformiswas connected to its ability to activate PlaC, whereas inN. lovaniensis, ProA appeared to have multiple functions. Together, these data document that the T2S system exports multiple effectors, including a novel one, which contribute in different ways to the broad host range ofL. pneumophila.

scholarly journals Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB ofLegionella pneumophilaContribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
J. Rasch ◽  
C. M. Ünal ◽  
A. Klages ◽  
Ü. Karsli ◽  
N. Heinsohn ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Temperature Tolerance ◽  
Lung Damage ◽  
Atypical Pneumonia ◽  
Content Type ◽  
Wide Range ◽  
Legionnaires Disease ◽  
Environmental Fitness ◽  
Sliding Motility

ABSTRACTThe gammaproteobacteriumLegionella pneumophilais the causative agent of Legionnaires’ disease, an atypical pneumonia that manifests itself with severe lung damage.L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. InL. pneumophilathe surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness ofL. pneumophila. Furthermore, they contribute to infection of the natural hostAcanthamoeba castellaniias well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators ofL. pneumophilawith compensatory action and pleiotropic effects.

scholarly journals First Staphylococcal Cassette ChromosomemecContaining amecB-Carrying Gene Complex Independent of Transposon Tn6045in a Macrococcus caseolyticus Isolate from a Canine Infection

2015 ◽  
Vol 59 (8) ◽  
pp. 4577-4583 ◽  
Author(s):  
Elena Gómez-Sanz ◽  
Sybille Schwendener ◽  
Andreas Thomann ◽  
Stefanie Gobeli Brawand ◽  
Vincent Perreten
Keyword(s):  
Integration Site ◽  
Direct Repeat ◽  
Open Reading Frames ◽  
Gene Complex ◽  
Direct Repeats ◽  
Content Type ◽  
Canine Infection ◽  
The Right ◽  
Lactamase Gene ◽  
Reading Frames

ABSTRACTA methicillin-resistantmecB-positiveMacrococcus caseolyticus(strain KM45013) was isolated from the nares of a dog with rhinitis. It contained a novel 39-kb transposon-defective completemecB-carrying staphylococcal cassette chromosomemecelement (SCCmecKM45013). SCCmecKM45013contained 49 coding sequences (CDSs), was integrated at the 3′ end of the chromosomalorfXgene, and was delimited at both ends by imperfect direct repeats functioning as integration site sequences (ISSs). SCCmecKM45013presented two discontinuous regions of homology (SCCmeccoverage of 35%) to the chromosomal and transposon Tn6045-associated SCCmec-like element ofM. caseolyticusJCSC7096: (i) themecgene complex (98.8% identity) and (ii) theccr-carrying segment (91.8% identity). Themecgene complex, located at the right junction of the cassette, also carried the β-lactamase geneblaZm(mecRm-mecIm-mecB-blaZm). SCCmecKM45013contained two cassette chromosome recombinase genes,ccrAm2andccrBm2, which shared 94.3% and 96.6% DNA identity with those of the SCCmec-like element of JCSC7096 but shared less than 52% DNA identity with the staphylococcalccrABandccrCgenes. Three distinct extrachromosomal circularized elements (the entire SCCmecKM45013, ΨSCCmecKM45013lacking theccrgenes, and SCCKM45013lackingmecB) flanked by one ISS copy, as well as the chromosomal regions remaining after excision, were detected. An unconventional circularized structure carrying themecBgene complex was associated with two extensive direct repeat regions, which enclosed two open reading frames (ORFs) (ORF46 and ORF51) flanking the chromosomalmecB-carrying gene complex. This study revealedM. caseolyticusas a potential disease-associated bacterium in dogs and also unveiled an SCCmecelement carryingmecBnot associated with Tn6045in the genusMacrococcus.

scholarly journals Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

2012 ◽  
Vol 78 (19) ◽  
pp. 6963-6974 ◽  
Author(s):  
Jose M. Quesada ◽  
María Isabel Soriano ◽  
Manuel Espinosa-Urgel
Keyword(s):  
Pseudomonas Putida ◽  
Genomic Island ◽  
Mobile Element ◽  
Direct Repeat ◽  
Promoter Sequence ◽  
Repeat Sequence ◽  
Genomic Islands ◽  
Pseudomonas Putida Kt2440 ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTThe stability of seven genomic islands ofPseudomonas putidaKT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location,attsites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.

scholarly journals A Unique cis -Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Giulia Oliva ◽  
Tobias Sahr ◽  
Monica Rolando ◽  
Maike Knoth ◽  
Carmen Buchrieser
Keyword(s):  
Life Cycle ◽  
Virulence Factors ◽  
Legionella Pneumophila ◽  
Noncoding Rna ◽  
Acanthamoeba Castellanii ◽  
Severe Pneumonia ◽  
Dependent Manner ◽  
Rnase Iii ◽  
Small Noncoding Rna ◽  
Cycle Dependent

ABSTRACT Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires’ disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis -encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5′ untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti- hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. IMPORTANCE The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq -encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated.

scholarly journals Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

2014 ◽  
Vol 83 (3) ◽  
pp. 1008-1018 ◽  
Author(s):  
Felizza F. Gunderson ◽  
Celeste A. Mallama ◽  
Stephanie G. Fairbairn ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Nuclease Activity ◽  
Infection Process ◽  
Host Cells ◽  
Mutant Form ◽  
Content Type ◽  
Intracellular Infection ◽  
Short Palindromic Repeat ◽  
Naegleria Lovaniensis

Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein ofL. pneumophilapromotes intracellular infection ofAcanthamoeba castellaniiandHartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated byL. pneumophilaCas2 appeared to be distinct from this function, becausecas2mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the othercasgenes were not impaired in infection ability. We now report that the Cas2 protein ofL. pneumophilahas both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain ofL. pneumophilathat naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, acas2mutant was impaired for infection ofWillaertia magnabut notNaegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

scholarly journals Wild ciliates differ in susceptibility to Legionella pneumophila JR32

Microbiology ◽  
2021 ◽  
Vol 167 (8) ◽  
Author(s):  
Airi Kawashiro ◽  
Torahiko Okubo ◽  
Shinji Nakamura ◽  
Jeewan Thapa ◽  
Masaki Miyake ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Deletion Mutant ◽  
Virulence Genes ◽  
Reference Strain ◽  
Virulence Gene ◽  
Cyst Formation ◽  
Harsh Environments ◽  
Content Type ◽  
Pellet Production ◽  
Low Sensitivity

We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Δicm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.

scholarly journals Legionella norrlandica sp. nov., isolated from the biopurification systems of wood processing plants

2015 ◽  
Vol 65 (Pt_2) ◽  
pp. 598-603 ◽  
Author(s):  
Kristina Rizzardi ◽  
Jadwiga Winiecka-Krusnell ◽  
Miriam Ramliden ◽  
Erik Alm ◽  
Sabina Andersson ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Legionella Pneumophila ◽  
Type Species ◽  
Sequence Similarity ◽  
Acanthamoeba Castellanii ◽  
Content Type ◽  
Wood Processing ◽  
Link Type ◽  
Processing Plants ◽  
Biopurification Systems

Fourteen isolates of an unknown species identified as belonging to the genus Legionella by selective growth on BCYE agar were isolated from the biopurification systems of three different wood processing plants. The mip gene sequence of all 14 isolates was identical and a close match alignment revealed 86 % sequence similarity with Legionella pneumophila serogroup 8. The whole genome of isolate LEGNT was sequenced, and a phylogenetic tree based on the alignment of 16S rRNA, mip, rpoB, rnpB and the 23S–5S intergenic region clustered LEGNT with L. pneumophila ATCC 33152T. Analysis of virulence factors showed that strain LEGNT carries the majority of known L. pneumophila virulence factors. An amoeba infection assay performed to assess the pathogenicity of strain LEGNT towards Acanthamoeba castellanii showed that it can establish a replication vacuole in A. castellanii but does not significantly affect replication of amoebae. Taken together, the results confirm that strain LEGNT represents a novel species of the genus Legionella , for which the name Legionella norrlandica sp. nov. is proposed. The type strain is LEGNT ( = ATCC BAA-2678T = CCUG 65936T).

scholarly journals LplR, a Repressor Belonging to the TetR Family, Regulates Expression of the l-Pantoyl Lactone Dehydrogenase Gene in Rhodococcus erythropolis

2012 ◽  
Vol 78 (22) ◽  
pp. 7923-7930 ◽  
Author(s):  
Dayong Si ◽  
Nobuyuki Urano ◽  
Sakayu Shimizu ◽  
Michihiko Kataoka
Keyword(s):  
Rhodococcus Erythropolis ◽  
Repeat Sequence ◽  
Open Reading Frames ◽  
Negative Regulator ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Recombinant Cells ◽  
Mobility Shift Assay ◽  
Multicopy Vector

ABSTRACTThel-pantoyl lactone (l-PL) dehydrogenase (LPLDH) gene (lpldh) has been cloned fromRhodococcus erythropolisAKU2103, and addition of 1,2-propanediol (1,2-PD) was shown to be required forlpldhexpression in this strain. In this study, based on an exploration of the nucleotide sequence aroundlpldh, a TetR-like regulator gene, which we designatedlplR, was found upstream oflpldh, and three putative open reading frames existed between the two genes. Disruption oflplRled to 22.8 times higherlpldhexpression, even without 1,2-PD induction, than that in wild-typeR. erythropolisAKU2103 without 1,2-PD addition. Introduction of a multicopy vector carryinglplR(multi-lplR) into the wild-type and ΔlplRstrains led to no detectable LPLDH activity even in the presence of 1,2-PD. The results of an electrophoretic mobility shift assay revealed that purified LplR bound to a 6-bp inverted-repeat sequence located in the promoter/operator region of the operon containinglpldh. These results indicated that LplR is a negative regulator inlpldhexpression. Based on the clarification of the expression mechanism oflpldh, recombinant cells showing high LPLDH activity were constructed and used as a catalyst for the conversion ofl-PL to ketopantoyl lactone. Finally, a promising production process ofd-PL fromdl-PL was constructed.

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