scholarly journals Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3569-3573 ◽  
Author(s):  
Claressa E. Lucas ◽  
Ellen Brown ◽  
Barry S. Fields
Keyword(s):  
Legionella Pneumophila ◽  
Wild Type Strain ◽  
Water Systems ◽  
Type Iv Pili ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Intracellular Replication ◽  
Type Iv

Legionellae colonize biofilms in building water systems, yet little is known about their interaction with the organisms in these microbial communities. The role of Legionella pneumophila type IV pili and the type II secretion pre-pilin peptidase was evaluated in a model biofilm system. L. pneumophila strains 130b (wild-type), BS100 (a type IV pili mutant) and NU243 (a pre-pilin peptidase mutant) were assessed for attachment and retention in an established biofilm. Strains 130b and NU243 colonized the biofilm at a similar level while BS100 attached at a tenfold lower level. Over time, NU243 dropped below the level of detection while BS100 remained in the biofilm throughout the course of the experiment. The wild-type strain decreased but remained at a considerably higher level than either of the mutants. Inclusion of amoebae with BS100 allowed for attachment and retention at a level similar to 130b. NU243, which displays reduced intracellular replication, was able to establish itself and persist in the presence of amoebae. Thus, type IV pili and the pre-pilin peptidase facilitate L. pneumophila colonization of biofilms but are not required in the presence of a host for intracellular replication.

scholarly journals Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation

2012 ◽  
Vol 86 (4) ◽  
pp. 805-818 ◽  
Author(s):  
David A. Cisneros ◽  
Gerard Pehau-Arnaudet ◽  
Olivera Francetic
Keyword(s):  
Secretion System ◽  
Type Iv Pili ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv ◽  
Type Ii Secretion System

scholarly journals icmT Is Essential for Pore Formation-Mediated Egress of Legionella pneumophila from Mammalian and Protozoan Cells

2002 ◽  
Vol 70 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Maelle Molmeret ◽  
O. A. Terry Alli ◽  
Steven Zink ◽  
Antje Flieger ◽  
Nicholas P. Cianciotto ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Type Strain ◽  
Pore Formation ◽  
Wild Type Strain ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Type Ii Secretion System

ABSTRACT The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.

scholarly journals Multidisciplinary Interrogation of a Crucial Protein Interface in the Type II Secretion System

2020 ◽  
Author(s):  
Cristian A Escobar ◽  
Badreddine Douzi ◽  
Geneviève Ball ◽  
Brice Barbat ◽  
Sebastien Alphonse ◽  
...  
Keyword(s):  
Structural Model ◽  
Secretion System ◽  
Type Iv Pili ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv ◽  
Type Ii Secretion System ◽  
Biophysical Techniques ◽  
Membrane Spanning

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The Type II secretion system (T2SS), a significant virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in understanding of the T2SS is the molecular role of a small 'pseudopilin' protein. Using multiple biophysical techniques, we have deciphered how this missing component of the Xcp T2SS architecture is structurally integrated, and thereby also unlocked its function. We demonstrate that the low abundance XcpH is the adapter that bridges a trimeric initiating tip complex XcpIJK with a periplasmic filament of XcpG subunits. Our model reveals that each pseudopilin protein caps an XcpG protofilament in an overall pseudopilus compatible with dimensions of the periplasm and the outer membrane-spanning secretin through which substrates of the T2SS pass. Unexpectedly, to fulfill its adapter function, the XcpH N-terminal helix must be unwound, a property shared with XcpG subunits. We provide the first complete structural model of a type IV filament, a result immediately transferable to understanding of other T2SS and the type IV pili.

scholarly journals Surface Translocation by Legionella pneumophila: a Form of Sliding Motility That Is Dependent upon Type II Protein Secretion

2008 ◽  
Vol 191 (5) ◽  
pp. 1537-1546 ◽  
Author(s):  
Catherine R. Stewart ◽  
Ombeline Rossier ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Type Iv Secretion ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Normal Surface ◽  
Type Iv ◽  
Plastic Surface ◽  
Sliding Motility ◽  
Surface Translocation

ABSTRACT Legionella pneumophila exhibits surface translocation when it is grown on a buffered charcoal yeast extract (BCYE) containing 0.5 to 1.0% agar. After 7 to 22 days of incubation, spreading legionellae appear in an amorphous, lobed pattern that is most manifest at 25 to 30°C. All nine L. pneumophila strains examined displayed the phenotype. Surface translocation was also exhibited by some, but not all, other Legionella species examined. L. pneumophila mutants that were lacking flagella and/or type IV pili behaved as the wild type did when plated on low-percentage agar, indicating that the surface translocation is not swarming or twitching motility. A translucent film was visible atop the BCYE agar, advancing ahead of the spreading legionellae. Based on its abilities to disperse water droplets and to promote the spreading of heterologous bacteria, the film appeared to manipulate surface tension and, as such, acted like a surfactant. Indeed, a sample obtained from the film rapidly dispersed when it was spotted onto a plastic surface. L. pneumophila type II secretion (Lsp) mutants, but not their complemented derivatives, were defective for both surface translocation and film production. In contrast, mutants defective for type IV secretion exhibited normal surface translocation. When lsp mutants were spotted onto film produced by the wild type, they were able to spread, suggesting that type II secretion promotes the elaboration of the Legionella surfactant. Together, these data indicate that L. pneumophila exhibits a form of surface translocation that is most akin to “sliding motility” and uniquely dependent upon type II secretion.

scholarly journals Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Life Cycles ◽  
Wild Type ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Reporter Systems ◽  
Brucella Neotomae

ABSTRACT We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

Type II Secretion and Type IV Pili of Francisella

2007 ◽  
Vol 1105 (1) ◽  
pp. 187-201 ◽  
Author(s):  
A. FORSBERG ◽  
T. GUINA
Keyword(s):  
Type Iv Pili ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv

scholarly journals The Dot/Icm Type IV Secretion System of Legionella pneumophila Is Essential for the Induction of Apoptosis in Human Macrophages

2002 ◽  
Vol 70 (3) ◽  
pp. 1657-1663 ◽  
Author(s):  
Steven D. Zink ◽  
Lisa Pedersen ◽  
Nicholas P. Cianciotto ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Bacterial Pathogen ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Ii Secretion ◽  
Type Iv ◽  
Induction Of Apoptosis

ABSTRACT We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.

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 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 Legionella pneumophila Catalase-Peroxidases Are Required for Proper Trafficking and Growth in Primary Macrophages

2003 ◽  
Vol 71 (8) ◽  
pp. 4526-4535 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Brenda Byrne ◽  
Yolande Chan ◽  
Michele S. Swanson ◽  
Howard M. Steinman
Keyword(s):  
Legionella Pneumophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Wild Type ◽  
Type Iv ◽  
Virulence Traits

ABSTRACT Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H2O2 compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.

Sign in / Sign up

Export Citation Format

Share Document