LcrG is Required for Efficient Translocation ofYersinia Yop Effector Proteins into Eukaryotic Cells

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance.

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Investigation of Bartonella quintana Host Immune Evasion

10.26686/wgtn.17131799.v1 ◽

2021 ◽

Author(s):

◽

Callum Lambert

Keyword(s):

Immune System ◽

Actin Cytoskeleton ◽

Host Cell ◽

Immune Evasion ◽

Mammalian Species ◽

Effector Proteins ◽

Host Cells ◽

Sand Fly ◽

Host Immune System ◽

Bartonella Quintana

<p>Bartonella is a genus of gram-negative alphaproteobacteria that infect mammals, causing both acute and chronic disease. Bartonella are re-emerging infectious pathogens that cause a variety of clinical syndromes in humans worldwide, including cat scratch disease, trench fever, bacillary angiomatosis, and endocarditis. Bartonella spp. are spread by biting arthropods such as the sand fly, cat flea, and body louse, and have been isolated from almost all mammalian species tested. Bartonella are a re-emerging concern as the number of confirmed Bartonella diagnoses are increasing, primarily in immunocompromised groups, homeless populations, refugee camps, and in veterinary workers. The three primary human disease-causing Bartonella spp. are B. henselae, B. quintana, and B. bacilliformis. Bartonella are known to subvert the host immune system and persist within the host, often causing bacteraemia which is difficult to effectively diagnose and treat. B. quintana infects humans; after introduction to the skin the bacteria implement numerous immune evasion mechanisms to enter the bloodstream and invade erythrocytes. The mechanisms by which B. quintana modulates and evades the immune system during early infection are almost entirely unknown. Following exposure to B. quintana, the bacteria encounter host immune cells but survive, evading these cells and disseminating into the lymphatic system and eventually bloodstream. This thesis project aimed to dissect the interactions between B. quintana and the human innate immune system to better understand the early stages of infection. A gentamicin protection assay was developed to investigate the ability of THP-1 macrophages, representing human macrophages present in the skin, to internalise B. quintana. These data revealed THP-1 cells were unable to effectively internalise B. quintana, although the mechanism responsible was not determined. Subsequent experiments investigated the role of the B. quintana Type IV secreted effector protein BepA1 in the inhibition of internalisation. Bacterial effector proteins often pathogenically modulate host cell signalling to benefit the bacteria, i.e., altering the actin cytoskeleton to inhibit phagocytosis or supressing immune responses. It was hypothesised BepA1 could play a role in inhibiting phagocytosis; therefore, the host cell target of BepA1 was investigated with a yeast two-hybrid system assay. The human protein Myozap was uncovered as a potential protein that interacts with BepA1. Myozap is expressed in cardiac and lung tissue as well as epithelial and endothelial cells, where it modulates Rho-dependent actin signalling, potentially affecting the actin cytoskeleton and the transcription factor MRTF-A, which influences immune reaction through modulation of NF-κB. To investigate the functional effects of BepA1 activity in host cells, HeLa cells were transfected with BepA1; cell migration and cytokine secretion were assessed, revealing a decrease in pro-inflammatory cytokines in BepA1-transfected cells in response to TNF-a stimulation. These data suggest BepA1 may be deployed by B. quintana during infection to suppress the host immune response and avoid clearance from the site of infection. This research addressed a major gap in our understanding of B. quintana infections. Improving our understanding of the interactions between Bartonella and the host immune system is an essential first step in the development of improved diagnostic techniques and treatments. </p>

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Host-adaptation in Legionellales is 2.4 Ga, coincident with eukaryogenesis

10.1101/852004 ◽

2019 ◽

Author(s):

Eric Hugoson ◽

Tea Ammunét ◽

Lionel Guy

Keyword(s):

Common Ancestor ◽

Gene Families ◽

Host Adaptation ◽

Effector Proteins ◽

Host Cells ◽

Eukaryotic Cells ◽

Last Eukaryotic Common Ancestor ◽

Molecular Machinery ◽

Type Ivb Secretion ◽

Bacterial Clade

AbstractBacteria adapting to living in a host cell caused the most salient events in the evolution of eukaryotes, namely the seminal fusion with an archaeon 1, and the emergence of both the mitochondrion and the chloroplast 2. A bacterial clade that may hold the key to understanding these events is the deep-branching gammaproteobacterial order Legionellales – containing among others Coxiella and Legionella – of which all known members grow inside eukaryotic cells 3. Here, by analyzing 35 novel Legionellales genomes mainly acquired through metagenomics, we show that this group is much more diverse than previously thought, and that key host-adaptation events took place very early in its evolution. Crucial virulence factors like the Type IVB secretion (Dot/Icm) system and two shared effector proteins were gained in the last Legionellales common ancestor (LLCA), while many metabolic gene families were lost in LLCA and its immediate descendants. We estimate that LLCA lived circa 2.4 Ga ago, predating the last eukaryotic common ancestor (LECA) by at least 0.5 Ga 4. These elements strongly indicate that host-adaptation arose only once in Legionellales, and that these bacteria were using advanced molecular machinery to exploit and manipulate host cells very early in eukaryogenesis.

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Investigation of Bartonella quintana Host Immune Evasion

10.26686/wgtn.17131799 ◽

2021 ◽

Author(s):

◽

Callum Lambert

Keyword(s):

Immune System ◽

Actin Cytoskeleton ◽

Host Cell ◽

Immune Evasion ◽

Mammalian Species ◽

Effector Proteins ◽

Host Cells ◽

Sand Fly ◽

Host Immune System ◽

Bartonella Quintana

<p>Bartonella is a genus of gram-negative alphaproteobacteria that infect mammals, causing both acute and chronic disease. Bartonella are re-emerging infectious pathogens that cause a variety of clinical syndromes in humans worldwide, including cat scratch disease, trench fever, bacillary angiomatosis, and endocarditis. Bartonella spp. are spread by biting arthropods such as the sand fly, cat flea, and body louse, and have been isolated from almost all mammalian species tested. Bartonella are a re-emerging concern as the number of confirmed Bartonella diagnoses are increasing, primarily in immunocompromised groups, homeless populations, refugee camps, and in veterinary workers. The three primary human disease-causing Bartonella spp. are B. henselae, B. quintana, and B. bacilliformis. Bartonella are known to subvert the host immune system and persist within the host, often causing bacteraemia which is difficult to effectively diagnose and treat. B. quintana infects humans; after introduction to the skin the bacteria implement numerous immune evasion mechanisms to enter the bloodstream and invade erythrocytes. The mechanisms by which B. quintana modulates and evades the immune system during early infection are almost entirely unknown. Following exposure to B. quintana, the bacteria encounter host immune cells but survive, evading these cells and disseminating into the lymphatic system and eventually bloodstream. This thesis project aimed to dissect the interactions between B. quintana and the human innate immune system to better understand the early stages of infection. A gentamicin protection assay was developed to investigate the ability of THP-1 macrophages, representing human macrophages present in the skin, to internalise B. quintana. These data revealed THP-1 cells were unable to effectively internalise B. quintana, although the mechanism responsible was not determined. Subsequent experiments investigated the role of the B. quintana Type IV secreted effector protein BepA1 in the inhibition of internalisation. Bacterial effector proteins often pathogenically modulate host cell signalling to benefit the bacteria, i.e., altering the actin cytoskeleton to inhibit phagocytosis or supressing immune responses. It was hypothesised BepA1 could play a role in inhibiting phagocytosis; therefore, the host cell target of BepA1 was investigated with a yeast two-hybrid system assay. The human protein Myozap was uncovered as a potential protein that interacts with BepA1. Myozap is expressed in cardiac and lung tissue as well as epithelial and endothelial cells, where it modulates Rho-dependent actin signalling, potentially affecting the actin cytoskeleton and the transcription factor MRTF-A, which influences immune reaction through modulation of NF-κB. To investigate the functional effects of BepA1 activity in host cells, HeLa cells were transfected with BepA1; cell migration and cytokine secretion were assessed, revealing a decrease in pro-inflammatory cytokines in BepA1-transfected cells in response to TNF-a stimulation. These data suggest BepA1 may be deployed by B. quintana during infection to suppress the host immune response and avoid clearance from the site of infection. This research addressed a major gap in our understanding of B. quintana infections. Improving our understanding of the interactions between Bartonella and the host immune system is an essential first step in the development of improved diagnostic techniques and treatments. </p>

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Gezielte Injektion von Effektoren durch Kontrolle der Proteindynamik

BIOspektrum ◽

10.1007/s12268-021-1667-4 ◽

2021 ◽

Vol 27 (7) ◽

pp. 697-700

Author(s):

Stephan Wimmi ◽

Florian Lindner ◽

Andreas Diepold

Keyword(s):

Yersinia Enterocolitica ◽

Type Iii Secretion ◽

Direct Injection ◽

Low Ph ◽

Secretion System ◽

Effector Proteins ◽

Eukaryotic Cells ◽

Site Of Action ◽

Rapid Activation ◽

Spatiotemporal Control

AbstractThe type III secretion system (T3SS) enables direct injection of bacterial effector proteins into eukaryotic cells. We found that the dynamic cytosolic interface of the system allows Yersinia enterocolitica to suppress premature secretion at low pH, ensuring rapid activation at the site of action. Exploiting this principle, we developed a light-controlled T3SS based on optogenetic interaction switches, which provides unprecedented spatiotemporal control of protein secretion and translocation.

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SUMO and SUMOylation Pathway at the Forefront of Host Immune Response

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.681057 ◽

2021 ◽

Vol 9 ◽

Author(s):

Sajeev T. K. ◽

Garima Joshi ◽

Pooja Arya ◽

Vibhuti Mahajan ◽

Akanksha Chaturvedi ◽

...

Keyword(s):

Immune Response ◽

Immune System ◽

Host Immune Response ◽

Defense Responses ◽

Effector Proteins ◽

Host Cells ◽

Host Immune System ◽

Target Proteins ◽

Adaptive Immune Cells ◽

Sumo Pathway

Pathogens pose a continuous challenge for the survival of the host species. In response to the pathogens, the host immune system mounts orchestrated defense responses initiating various mechanisms both at the cellular and molecular levels, including multiple post-translational modifications (PTMs) leading to the initiation of signaling pathways. The network of such pathways results in the recruitment of various innate immune components and cells at the site of infection and activation of the adaptive immune cells, which work in synergy to combat the pathogens. Ubiquitination is one of the most commonly used PTMs. Host cells utilize ubiquitination for both temporal and spatial regulation of immune response pathways. Over the last decade, ubiquitin family proteins, particularly small ubiquitin-related modifiers (SUMO), have been widely implicated in host immune response. SUMOs are ubiquitin-like (Ubl) proteins transiently conjugated to a wide variety of proteins through SUMOylation. SUMOs primarily exert their effect on target proteins by covalently modifying them. However, SUMO also engages in a non-covalent interaction with the SUMO-interacting motif (SIM) in target proteins. Unlike ubiquitination, SUMOylation alters localization, interactions, functions, or stability of target proteins. This review provides an overview of the interplay of SUMOylation and immune signaling and development pathways in general. Additionally, we discuss in detail the regulation exerted by covalent SUMO modifications of target proteins, and SIM mediated non-covalent interactions with several effector proteins. In addition, we provide a comprehensive review of the literature on the importance of the SUMO pathway in the development and maintenance of a robust immune system network of the host. We also summarize how pathogens modulate the host SUMO cycle to sustain infectability. Studies dealing mainly with SUMO pathway proteins in the immune system are still in infancy. We anticipate that the field will see a thorough and more directed analysis of the SUMO pathway in regulating different cells and pathways of the immune system. Our current understanding of the importance of the SUMO pathway in the immune system necessitates an urgent need to synthesize specific inhibitors, bioactive regulatory molecules, as novel therapeutic targets.

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Roles of LcrG and LcrV during Type III Targeting of Effector Yops by Yersinia enterocolitica

Journal of Bacteriology ◽

10.1128/jb.183.15.4588-4598.2001 ◽

2001 ◽

Vol 183 (15) ◽

pp. 4588-4598 ◽

Cited By ~ 50

Author(s):

Kristin L. DeBord ◽

Vincent T. Lee ◽

Olaf Schneewind

Keyword(s):

Plasma Membrane ◽

Yersinia Enterocolitica ◽

Negative Regulator ◽

Host Cells ◽

Eukaryotic Cells ◽

Extracellular Medium ◽

Bacterial Surface ◽

Type Iii ◽

Surface Receptor ◽

Bacterial Cytoplasm

ABSTRACT Yersinia enterocolitica target effector Yop proteins into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. LcrG and LcrV have been suggested to fulfill essential functions during the type III targeting of effector Yops. It is reported here that knockout mutations of lcrG caused mutant yersiniae to prematurely secrete Yops into the extracellular medium without abolishing the type III targeting mechanism (Los phenotype [loss of type III targeting specificity]). Knockout mutations inlcrV reduced type III targeting of mutant yersiniae but did not promote secretion into the extracellular medium (Not [no type III targeting]). However, knockout mutations in both genes caused ΔlcrGV yersiniae to display a Los phenotype similar to that of strains carrying knockout mutations in lcrG alone. LcrG binding to LcrV resulted in the formation of soluble LcrGV complexes in the bacterial cytoplasm. Membrane-associated, bacterial-surface-displayed or -secreted LcrG could not be detected. Most of LcrV was located in the bacterial cytoplasm; however, small amounts were secreted into the extracellular medium. These data support a model whereby LcrG may act as a negative regulator of type III targeting in the bacterial cytoplasm, an activity that is modulated by LcrG binding to LcrV. No support could be gathered for the hypothesis whereby LcrG and LcrV may act as a bacterial surface receptor for host cells, allowing effector Yop translocation across the eukaryotic plasma membrane.

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Adaptive evolution among cytoplasmic piRNA proteins leads to decreased genomic auto-immunity

10.1101/521773 ◽

2019 ◽

Cited By ~ 1

Author(s):

Luyang Wang ◽

Daniel A. Barbash ◽

Erin S. Kelleher

Keyword(s):

Immune System ◽

Transposable Elements ◽

Adaptive Evolution ◽

Small Rna ◽

Functional Divergence ◽

Harmful Effect ◽

Effector Proteins ◽

Host Cells ◽

Selfish Dna ◽

Pirna Pathway

AbstractIn metazoan germlines, the piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). In response to dynamic changes in TE content, host genomes are proposed to alter the piRNAs that they produce in order to silence the most active TE families. Surprisingly, however, piRNA pathway proteins, which execute piRNA biogenesis and enforce silencing of targeted sequences, also evolve rapidly and adaptively in animals. If TE silencing evolves through changes in piRNAs, what necessitates changes in piRNA pathway proteins? Here we used interspecific complementation to test for functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. Surprisingly, we observed interspecific divergence in the regulation of only a handful of TE families, which were more robustly silenced by the heterospecific piRNA pathway protein. This suggests that positive selection does not act on piRNA effector proteins to enhance their function in TE repression, but rather that TEs may evolve to “escape” silencing by homospecific alleles. We also discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, suggesting the avoidance of genomic auto-immunity as a critical target of selection. Our observations suggest that piRNA effector proteins are subject to an evolutionary trade-off between defending the host genome from the harmful effect of TEs while also minimizing friendly fire against host genes.Author SummaryTransposable elements are mobile fragments of selfish DNA that burden host genomes with deleterious mutations and incite genome instability. Host cells employ a specialized small-RNA mediated silencing pathway, the piRNA pathway, to act as a genomic immune system suppressing the mobilization of TEs. Changes in genomic TE content are met with rapid changes in the piRNA pool, thereby maintain host control over transposition. However, piRNA pathway proteins—which enact piRNA biogenesis and silence target TEs—also evolve adaptively. To isolate forces that underlie this adaptive evolution, we examined functional divergence between two Drosophila species for three adaptively evolving piRNA pathway proteins. To our surprise, we found very few differences in TE regulation, suggesting that evolution has not generally acted to enhance control of TE parasites. Rather, we discovered interspecific differences in the regulation of host mRNAs for two proteins, which suggested that proteins evolve to avoid off-target silencing of host transcripts. We propose that the avoidance of such “genomic autoimmunity” is an important and underappreciated force driving the adaptive evolution of piRNA proteins.

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Live imaging of Yersinia translocon formation and immune recognition in host cells

10.1101/2022.01.10.475601 ◽

2022 ◽

Author(s):

Maren Rudolph ◽

Alexander Carsten ◽

Martin Aepfelbacher ◽

Manuel Wolters

Keyword(s):

Immune System ◽

Membrane Damage ◽

Effector Proteins ◽

Host Cells ◽

Immune Recognition ◽

Type Three Secretion System ◽

Galectin 3 ◽

Infected Cells ◽

Host Cell Membranes ◽

Phagosomal Membrane

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia -containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

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Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

Nature Communications ◽

10.1038/s41467-021-24277-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Plinio S. Vieira ◽

Isabela M. Bonfim ◽

Evandro A. Araujo ◽

Ricardo R. Melo ◽

Augusto R. Lima ◽

...

Keyword(s):

Virulence Factors ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Model Organism ◽

Citrus Canker ◽

Effector Proteins ◽

Glycoside Hydrolases ◽

Host Cells ◽

System A ◽

Enzymatic Machinery

AbstractXyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

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