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 The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2732-2744 ◽  
Author(s):  
Jessica Y. Tyson ◽  
Paloma Vargas ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Type Ii Secretion ◽  
The Novel ◽  
Type Ii ◽  
Intracellular Infection ◽  
Novel Proteins ◽  
Hartmannella Vermiformis ◽  
The Individual ◽  
Naegleria Lovaniensis

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires’ disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.

scholarly journals Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

2016 ◽  
Vol 84 (12) ◽  
pp. 3313-3327 ◽  
Author(s):  
Richard C. White ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Host Cells ◽  
Growth Defect ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Murine Bone Marrow ◽  
Intracellular Infection ◽  
Growth Difference

Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages byLegionella pneumophila. In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in theLegionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, asidM lspFdouble mutant had an intracellular growth defect that was more dramatic than that of thelspFmutant (and asidMmutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.

scholarly journals Legionella pneumophila Type II Secretion Dampens the Cytokine Response of Infected Macrophages and Epithelia

2011 ◽  
Vol 79 (5) ◽  
pp. 1984-1997 ◽  
Author(s):  
Kessler McCoy-Simandle ◽  
Catherine R. Stewart ◽  
Jenny Dao ◽  
Sruti DebRoy ◽  
Ombeline Rossier ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Host Cells ◽  
Infected Host ◽  
Pcr Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Intracellular Infection ◽  
The Impact

ABSTRACTThe type II secretion (T2S) system ofLegionella pneumophilais required for the ability of the bacterium to grow within the lungs of A/J mice. By utilizing mutants lacking T2S (lsp), we now document that T2S promotes the intracellular infection of both multiple types of macrophages and lung epithelia. Following infection of macrophages,lspmutants (but not a complemented mutant) elicited significantly higher levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-10, IL-8, IL-1β, and MCP-1 within tissue culture supernatants. A similar result was obtained with infected lung epithelial cell lines and the lungs of infected A/J mice. Infection with a mutant specifically lacking the T2S-dependent ProA protease (but not a complementedproAmutant) resulted in partial elevation of cytokine levels. These data demonstrate that the T2S system ofL. pneumophiladampens the cytokine/chemokine output of infected host cells. Upon quantitative reverse transcription (RT)-PCR analysis of infected host cells, anlspFmutant, but not theproAmutant, produced significantly higher levels of cytokine transcripts, implying that some T2S-dependent effectors dampen signal transduction and transcription but that others, such as ProA, act at a posttranscriptional step in cytokine expression. In summary, the impact of T2S on lung infection is a combination of at least three factors: the promotion of growth in macrophages, the facilitation of growth in epithelia, and the dampening of the chemokine and cytokine output from infected host cells. To our knowledge, these data are the first to identify a link between a T2S system and the modulation of immune factors following intracellular infection.

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 Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Richard C. White ◽  
Hilary K. Truchan ◽  
Huaixin Zheng ◽  
Jessica Y. Tyson ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Recent Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Macrophage Infection ◽  
Content Type ◽  
Intracellular Infection

ABSTRACT It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii. Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant’s defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

scholarly journals Expanding Role of Type II Secretion in Bacterial Pathogenesis and Beyond

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Nicholas P. Cianciotto ◽  
Richard C. White
Keyword(s):  
Legionella Pneumophila ◽  
Xanthomonas Campestris ◽  
Plant Pathogens ◽  
Host Cells ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Tissue Destruction ◽  
Dickeya Dadantii ◽  
Content Type ◽  
Animal Pathogens

ABSTRACT Type II secretion (T2S) is one means by which Gram-negative pathogens secrete proteins into the extracellular milieu and/or host organisms. Based upon recent genome sequencing, it is clear that T2S is largely restricted to the Proteobacteria, occurring in many, but not all, genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria classes. Prominent human and/or animal pathogens that express a T2S system(s) include Acinetobacter baumannii, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, and Yersinia enterocolitica. T2S-expressing plant pathogens include Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa. T2S also occurs in nonpathogenic bacteria, facilitating symbioses, among other things. The output of a T2S system can range from only one to dozens of secreted proteins, encompassing a diverse array of toxins, degradative enzymes, and other effectors, including novel proteins. Pathogenic processes mediated by T2S include the death of host cells, degradation of tissue, suppression of innate immunity, adherence to host surfaces, biofilm formation, invasion into and growth within host cells, nutrient assimilation, and alterations in host ion flux. The reach of T2S is perhaps best illustrated by those bacteria that clearly use it for both environmental survival and virulence; e.g., L. pneumophila employs T2S for infection of amoebae, growth within lung cells, dampening of cytokines, and tissue destruction. This minireview provides an update on the types of bacteria that have T2S, the kinds of proteins that are secreted via T2S, and how T2S substrates promote infection.

scholarly journals The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Celeste A. Mallama ◽  
Kessler McCoy-Simandle ◽  
Nicholas P. Cianciotto
Keyword(s):  
Protein Kinase ◽  
Legionella Pneumophila ◽  
Mitogen Activated Protein Kinase ◽  
Human Macrophage ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Toll Like Receptor ◽  
Content Type ◽  
Human Macrophages ◽  
Toll Like Receptor 2

ABSTRACT Previously, we reported that mutants of Legionella pneumophila lacking a type II secretion (T2S) system elicit higher levels of cytokines (e.g., interleukin-6 [IL-6]) following infection of U937 cells, a human macrophage-like cell line. We now show that this effect of T2S is also manifest upon infection of human THP-1 macrophages and peripheral blood monocytes but does not occur during infection of murine macrophages. Supporting the hypothesis that T2S acts to dampen the triggering of an innate immune response, we observed that the mitogen-activated protein kinase (MAPK) and nuclear transcription factor kappa B (NF-κB) pathways are more highly stimulated upon infection with the T2S mutant than upon infection with the wild type. By using short hairpin RNA to deplete proteins involved in specific pathogen-associated molecular pattern (PAMP) recognition pathways, we determined that the dampening effect of the T2S system was not dependent on nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible protein I (RIG-I)-like receptors (RLRs), double-stranded RNA (dsRNA)-dependent protein kinase receptor (PKR), or TIR domain-containing adaptor inducing interferon beta (TRIF) signaling or an apoptosis-associated speck-like protein containing a CARD (ASC)- or caspase-4-dependent inflammasome. However, the dampening effect of T2S on IL-6 production was significantly reduced upon gene knockdown of myeloid differentiation primary response 88 (MyD88), TANK binding kinase 1 (TBK1), or Toll-like receptor 2 (TLR2). These data indicate that the L. pneumophila T2S system dampens the signaling of the TLR2 pathway in infected human macrophages. We also document the importance of PKR, TRIF, and TBK1 in cytokine secretion during L. pneumophila infection of macrophages.

scholarly journals Functional Characterization of EpsC, a Component of the Type II Secretion System, in the Pathogenicity of Vibrio vulnificus

2011 ◽  
Vol 79 (10) ◽  
pp. 4068-4080 ◽  
Author(s):  
Won Hwang ◽  
Na Yeon Lee ◽  
Juri Kim ◽  
Mi-Ae Lee ◽  
Kun-Soo Kim ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Secretion System ◽  
Host Cells ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Secreted Factors

ABSTRACTEpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium,Vibrio vulnificus, to evaluate its role in eliciting virulence. AnespC-deleted mutant ofV. vulnificusdisplayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by intranscomplementation of the intactepsCgene. Analyses of cellular fractions revealed that VvhA and VvpE of theΔepsCmutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of aV. vulnificusmutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin ofV. vulnificus, RtxA. Therefore, the T2SS is required forV. vulnificuspathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.

scholarly journals Type II Protein Secretion Is a Subset of the PilD-Dependent Processes That Facilitate Intracellular Infection byLegionella pneumophila

2001 ◽  
Vol 69 (4) ◽  
pp. 2092-2098 ◽  
Author(s):  
Ombeline Rossier ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Enzymatic Activities ◽  
Colony Morphology ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Intracellular Infection ◽  
Type Iv ◽  
Fold Reduction ◽  
Intracellular Multiplication ◽  
Prepilin Peptidase

ABSTRACT Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pilEL ) mutant. To determine whether thesepilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, orlspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilEL strains, bothlspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of thepilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, bothlsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, thepilD mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promotingL. pneumophila intracellular infection of human cells.

scholarly journals A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 882-890 ◽  
Author(s):  
Ombeline Rossier ◽  
Jenny Dao ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Mutational Analysis ◽  
Critical Role ◽  
Rnase Activity ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Intracellular Infection ◽  
Zinc Metalloprotease ◽  
Type Ii Secretion System ◽  
Hartmannella Vermiformis

Type II protein secretion plays a role in a wide variety of functions that are important for the ecology and pathogenesis of Legionella pneumophila. Perhaps most dramatic is the critical role that this secretion pathway has in L. pneumophila intracellular infection of aquatic protozoa. Recently, we showed that virulent L. pneumophila strain 130b secretes RNase activity through its type II secretion system. We now report the cloning and mutational analysis of the gene (srnA) encoding that novel type of secreted activity. The SrnA protein was defined as being a member of the T2 family of secreted RNases. Supernatants from mutants inactivated for srnA completely lacked RNase activity, indicating that SrnA is the major secreted RNase of L. pneumophila. Although srnA mutants grew normally in bacteriological media and human U937 cell macrophages, they were impaired in their ability to grow within Hartmannella vermiformis amoebae. This finding represents the second identification of a L. pneumophila type II effector being necessary for optimal intracellular infection of amoebae, with the first being the ProA zinc metalloprotease. Newly constructed srnA proA double mutants displayed an even larger infection defect that appeared to be the additive result of losing both SrnA and ProA. Overall, these data represent the first demonstration of a secreted RNase promoting an intracellular infection event, and support our long-standing hypothesis that the infection defects of L. pneumophila type II secretion mutants are due to the loss of multiple secreted effectors.

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