Legionella pneumophila Entry GenertxA Is Involved in Virulence

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.

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

Infection and Immunity ◽

10.1128/iai.69.4.2116-2122.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2116-2122 ◽

Cited By ~ 85

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.

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Legionella pneumophila — The causative agent of Legionnaires’ disease

Progress In Microbes & Molecular Biology ◽

10.36877/pmmb.a0000193 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Loh Teng Hern Tan ◽

Wei Yu Tee ◽

Tahir Mehmood Khan ◽

Long Chiau Ming ◽

Vengadesh Letchumanan

Keyword(s):

Causative Agent ◽

Legionella Pneumophila ◽

Current Knowledge ◽

Host Cells ◽

Intracellular Pathogen ◽

Respiratory Pathogens ◽

Appropriate Treatment ◽

Global Epidemiology ◽

Early Testing ◽

Legionnaires Disease

Over the years, Legionella pneumophila has increasingly become a public health threat that causes sporadic and epidemic community-acquired and nosocomial-acquired pneumonia. Thus, this review aims to discuss the current knowledge of L. pneumophila, focusing on the global epidemiology, clinical features, diagnosis and treatment of Legionnaires’ disease (LD). Legionella bacteria are Gram-negative rod-shaped bacteria that are ubiquitous in aquatic environments. L. pneumophila was first discovered in 1976 and recognized as the causative agent of LD. L. pneumophila is a facultative intracellular pathogen that infects and replicates within eukaryotic host cells such as macrophages and protozoan. Diagnosis of LD remains a significant challenge as the clinical manifestation of LD is hardly distinguishable from pneumonia caused by other respiratory pathogens. Therefore, early testing and appropriate treatment are keys to alleviating the rising morbidity and mortality caused by LD.

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Deletion of oxyR in Legionella pneumophila causes growth defect on agar

Canadian Journal of Microbiology ◽

10.1139/cjm-2018-0129 ◽

2018 ◽

Vol 64 (12) ◽

pp. 1030-1041 ◽

Cited By ~ 1

Author(s):

Nilmini Mendis ◽

Hana Trigui ◽

Mariam Saad ◽

Adrianna Tsang ◽

Sébastien P. Faucher

Keyword(s):

Legionella Pneumophila ◽

Water Environment ◽

Host Cells ◽

Growth Defect ◽

Intracellular Pathogen ◽

Wild Type ◽

Severe Growth Defect ◽

In Trans ◽

Severe Growth

The intracellular pathogen Legionella pneumophila (Lp) is a strict aerobe, surviving and replicating in environments where it frequently encounters reactive oxygen species (ROS), such as the nutrient-poor water environment and its replicative niche inside host cells. In many proteobacteria, the LysR-type regulator OxyR controls the oxidative stress response; however, the importance of the OxyR homologue in Lp is still unclear. Therefore, we undertook the characterization of phenotypes associated with the deletion of oxyR in Lp. Contrary to the wild type, the oxyR deletion mutant exhibits a severe growth defect on charcoal – yeast extract (CYE) agar lacking α-ketoglutarate supplementation. Growth in AYE broth (CYE without agar and charcoal), in amoeba and in human cultured macrophages, and survival in water is unaffected by the deletion. Supplementing CYE agar with antioxidants that neutralize ROS or introducing the oxyR gene in trans rescues the observed growth defect. Moreover, the mutant grows as well as the wild type on CYE plates made with agarose instead of agar, suggesting that a compound present in the latter is responsible for the growth defect phenotype.

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Cyclic Diguanylate Signaling Proteins Control Intracellular Growth of Legionella pneumophila

mBio ◽

10.1128/mbio.00316-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 31

Author(s):

Assaf Levi ◽

Marc Folcher ◽

Urs Jenal ◽

Howard A. Shuman

Keyword(s):

Causative Agent ◽

Legionella Pneumophila ◽

Second Messenger ◽

Host Cells ◽

Diguanylate Cyclase ◽

Intracellular Growth ◽

Cyclic Diguanylate ◽

Content Type ◽

Legionnaires Disease

ABSTRACTProteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle ofLegionella pneumophila, the causative agent of Legionnaires’ disease, has not previously been examined. TheL. pneumophilagenome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes ascdgS(cyclicdiguanylatesignaling) genes. Strains ofL. pneumophilacontaining deletions of all individualcdgSgenes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, severalcdgSgenes strongly decreased the ability ofL. pneumophilato grow inside host cells. Expression of thesecdgSgenes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth inL. pneumophila.L. pneumophilastrains overexpressing thesecdgSgenes were less cytotoxic to THP-1 macrophages than wild-typeL. pneumophilabut retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused bycdgSgene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of thecdgSgenes in aSalmonella entericaserovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that severalcdgSgenes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability ofL. pneumophilato grow in host cells.IMPORTANCEAll bacteria must sense and respond to environmental cues. Intracellular bacterial pathogens must detect and respond to host functions that limit their ability to carry out a successful infection. Small-molecule second messengers play key roles in transmitting signals from environmental receptors to the proteins and other components that respond to signals. Cyclic diguanylate is a ubiquitous bacterial second messenger known to play an important role in many sensing and signaling systems in bacteria. The causative agent of Legionnaires’ disease,Legionella pneumophila, is an intracellular pathogen that grows inside environmental protists and human macrophages by subverting the normal processes that these cells use to capture and destroy bacteria. We show that the several cyclic diguanylate signaling components inLegionellaplay a role in the ability to grow inside both kinds of host cells. This work highlights the role of cyclic diguanylate signaling during intracellular growth.

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Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

Infection and Immunity ◽

10.1128/iai.72.10.5983-5992.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5983-5992 ◽

Cited By ~ 56

Author(s):

Jessica A. Sexton ◽

Jennifer L. Miller ◽

Aki Yoneda ◽

Thomas E. Kehl-Fie ◽

Joseph P. Vogel

Keyword(s):

Cell Surface ◽

Legionella Pneumophila ◽

Bacterial Species ◽

Wide Distribution ◽

Host Cells ◽

Growth Defect ◽

Intracellular Growth ◽

Homologous Proteins ◽

Type Iv ◽

Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

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hfqPlays Important Roles in Virulence and Stress Adaptation in Cronobacter sakazakii ATCC 29544

Infection and Immunity ◽

10.1128/iai.03161-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 2089-2098 ◽

Cited By ~ 21

Author(s):

Seongok Kim ◽

Hyelyeon Hwang ◽

Kwang-Pyo Kim ◽

Hyunjin Yoon ◽

Dong-Hyun Kang ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Species ◽

Soft Agar ◽

Host Cells ◽

Neonatal Meningitis ◽

Animal Cells ◽

Content Type ◽

Flagellar Biosynthesis

Cronobacterspp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated withCronobacterinfection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq inC. sakazakiivirulence. In the absence ofhfq,C. sakazakiiwas highly attenuated in disseminationin vivo, showed defects in invasion (3-fold) into animal cells and survival (103-fold) within host cells, and exhibited low resistance to hydrogen peroxide (102-fold). Remarkably, the loss ofhfqled to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lackinghfq. Together, these data strongly suggest thathfqplays important roles in the virulence ofC. sakazakiiby participating in the regulation of multiple genes.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00467-21 ◽

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.

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Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

Journal of Cell Science ◽

10.1242/jcs.115.13.2669 ◽

2002 ◽

Vol 115 (13) ◽

pp. 2669-2678 ◽

Cited By ~ 2

Author(s):

Anna Gustavsson ◽

Annika Armulik ◽

Cord Brakebusch ◽

Reinhard Fässler ◽

Staffan Johansson ◽

...

Keyword(s):

Marked Reduction ◽

Yersinia Pseudotuberculosis ◽

Host Cells ◽

Β1 Integrin ◽

Complex Structures ◽

Wild Type ◽

Similar Reduction ◽

Focal Complex ◽

Β1 Integrins

Invasin of Yersinia pseudotuberculosis binds to β1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the β1-integrin-mediated internalization of Yersinia, a β1-integrin-deficient cell line, GD25, transfected with wild-type β1A, β1B or different mutants of the β1A subunit was used. Both β1A and β1B bound to invasin-expressing bacteria, but only β1A was able to mediate internalization of the bacteria. The cytoplasmic region of β1A, differing from β1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

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The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

Infection and Immunity ◽

10.1128/iai.00261-21 ◽

2021 ◽

Author(s):

Luying Liu ◽

Craig R. Roy

Keyword(s):

Legionella Pneumophila ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Effector Protein ◽

Intracellular Replication ◽

Phagocytic Cells ◽

Host Molecules ◽

Type Iv ◽

Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

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Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

Infection and Immunity ◽

10.1128/iai.00841-19 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

Susmita Ghosh ◽

Elizabeth A. Ruelke ◽

Joshua C. Ferrell ◽

Maria D. Bodero ◽

Kenneth A. Fields ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Host Cell ◽

Host Cells ◽

Actin Binding ◽

Wild Type ◽

Content Type ◽

Allelic Exchange ◽

Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

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