scholarly journals Orientia tsutsugamushi Nucleomodulin Ank13 Exploits the RaDAR Nuclear Import Pathway To Modulate Host Cell Transcription

mBio ◽  
2021 ◽  
Author(s):  
Haley E. Adcox ◽  
Amanda L. Hatke ◽  
Shelby E. Andersen ◽  
Sarika Gupta ◽  
Nathan B. Otto ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Scrub Typhus ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Orientia Tsutsugamushi ◽  
Microbial Protein ◽  
Content Type ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene

Nucleomodulins are recently defined effectors used by diverse intracellular bacteria to manipulate eukaryotic gene expression and convert host cells into hospitable niches. How nucleomodulins enter the nucleus, their functional domains, and the genes that they modulate are incompletely characterized. Orientia tsutsugamushi is an intracellular bacterial pathogen that causes scrub typhus, which can be fatal. O. tsutsugamushi Ank13 is the first example of a microbial protein that coopts eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import.

scholarly journals Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

2015 ◽  
Vol 197 (19) ◽  
pp. 3097-3109 ◽  
Author(s):  
Andrea R. Beyer ◽  
Lauren VieBrock ◽  
Kyle G. Rodino ◽  
Daniel P. Miller ◽  
Brittney K. Tegels ◽  
...  
Keyword(s):  
Host Cell ◽  
Ubiquitin Ligase ◽  
Scrub Typhus ◽  
Ankyrin Repeat ◽  
Intracellular Bacterium ◽  
Host Cells ◽  
Orientia Tsutsugamushi ◽  
Ligand Interaction ◽  
Content Type ◽  
Cell Functions

ABSTRACTA rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions.Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here,in silicoand manual sequence analyses revealed that a large proportion ofO. tsutsugamushistrain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathioneS-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitatedO. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. AligningO. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate thatO. tsutsugamushistrain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that anO. tsutsugamushiAnk does so during infection, and advance overall understanding of microbial F-box proteins.IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets.Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates thatO. tsutsugamushistrain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that anOrientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fideOrientiaeffector and the first example of an endogenous F-box-containing Ank–mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

scholarly journals Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Nicholas A. Wood ◽  
Krystal Y. Chung ◽  
Amanda M. Blocker ◽  
Nathalia Rodrigues de Almeida ◽  
Martin Conda-Sheridan ◽  
...  
Keyword(s):  
Host Cells ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Clp Protease ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Developmental Form ◽  
Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

scholarly journals Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen

2016 ◽  
Vol 84 (4) ◽  
pp. 1083-1091 ◽  
Author(s):  
Ryan McCormack ◽  
Wael Bahnan ◽  
Niraj Shrestha ◽  
Justin Boucher ◽  
Marcella Barreto ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Virulence Gene ◽  
Systemic Infection ◽  
Protective Role ◽  
Host Cells ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Membrane Bound ◽  
High Level ◽  
Macpf Domain

The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1,Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogenListeria monocytogenes. Within a few hours of systemic infection, the massive proliferation ofL. monocytogenesinPerforin-2−/−mice leads to a rapid appearance of acute disease symptoms. We go on to show in culturedPerforin-2−/−cells that the vacuole-to-cytosol transitioning ofL. monocytogenesis greatly accelerated. Unexpectedly, we found that inPerforin-2−/−macrophages,Listeria-containing vacuoles quickly (≤15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation ofL. monocytogenesto its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenesstrain expressing virulence factors at a constitutively high level replicated equally well inPerforin-2+/+andPerforin-2−/−macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification ofListeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.

scholarly journals Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Jin-Hahn Kim ◽  
Akhilesh Kumar Chaurasia ◽  
Nayab Batool ◽  
Kwan Soo Ko ◽  
Kyeong Kyu Kim
Keyword(s):  
Host Cells ◽  
Intracellular Bacteria ◽  
Protection Assay ◽  
Host Cell Membrane ◽  
Bacterial Killing ◽  
Content Type ◽  
Gentamicin Protection Assay ◽  
Enzyme Protection ◽  
The Difference ◽  
Kinetics Of

ABSTRACTPrecise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellularStaphylococcus aureus. We found that the difference between the two methods for the recovery of intracellular CFU ofS. aureuswas about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

scholarly journals Prospective Evaluation of Commercial Antibody-Based Rapid Tests in Combination with a Loop-Mediated Isothermal Amplification PCR Assay for Detection of Orientia tsutsugamushi during the Acute Phase of Scrub Typhus Infection

2012 ◽  
Vol 19 (3) ◽  
pp. 391-395 ◽  
Author(s):  
Stuart D. Blacksell ◽  
Daniel H. Paris ◽  
Wirongrong Chierakul ◽  
Vanaporn Wuthiekanun ◽  
Achara Teeratakul ◽  
...  
Keyword(s):  
Antibody Titer ◽  
Scrub Typhus ◽  
Orientia Tsutsugamushi ◽  
Pcr Assay ◽  
Content Type ◽  
Igm Antibody ◽  
Rapid Tests ◽  
Increased Sensitivity ◽  
Minimal Reduction ◽  
Diagnostic Capabilities

ABSTRACTSamples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection ofOrientia tsutsugamushiIgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity.

scholarly journals Kinetics and Magnitude of Antibody Responses against the Conserved 47-Kilodalton Antigen and the Variable 56-Kilodalton Antigen in Scrub Typhus Patients

2011 ◽  
Vol 18 (6) ◽  
pp. 1021-1027 ◽  
Author(s):  
Hua-Wei Chen ◽  
Zhiwen Zhang ◽  
Erin Huber ◽  
Elissa Mutumanje ◽  
Chien-Chung Chao ◽  
...  
Keyword(s):  
Scrub Typhus ◽  
Secondary Infection ◽  
Systematic Investigation ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Orientia Tsutsugamushi ◽  
Protein Antigens ◽  
Content Type ◽  
Molecular Sizes ◽  
Kinetics Of

ABSTRACTWestern blot analysis ofOrientia tsutsugamushiwhole-cell lysates with scrub typhus patient sera has identified at least five protein antigens ofO. tsutsugamushiwith molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.

An inducible expression system for recombinant Sca proteins with an autotransporter domain from Orientia tsutsugamushi in Escherichia coli

2020 ◽  
Vol 27 ◽  
Author(s):  
Hyejin Cho ◽  
Kwang-Sun Kim
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Scrub Typhus ◽  
Expression System ◽  
Protein Yield ◽  
Host Cells ◽  
Dilution Method ◽  
Orientia Tsutsugamushi ◽  
Dna Encoding ◽  
Protein Fragments

Background: Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative bacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization of the Ot strain into host cells and are suitable resources for vaccine development and tools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter (AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be promising candidates for vaccine development. These proteins are typically large and contain inhibitory domains; therefore, recombinant proteins without such domains have been evaluated for this purpose. However, the expression for recombinant Sca proteins containing the AT domain, which might largely affect their protective role against scrub typhus, has not been analyzed and optimized. Objective: In this study, we optimized individual genes encoding Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] harboring the AT domain. Methods: To this end we subcloned sequences of codon-optimized DNA encoding Sca protein fragments into the Escherichia coli expression vector. In addition, the expression condition for individual Sca fragments was optimized, and the proteins were purified using one-step histidine-tag column method. The purified proteins were re-folded by serial dilution method, followed by BCA quantification and densitometric analysis to estimate the protein yield and purity. Results: We prepared platforms for expression of recombinant Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] containing an AT domain but no signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with >70% of purity was ScaC (257–576), ScaE (35–760), ScaD (26-998), and ScaA (27- 1461) in order. Conclusion: Our results could be used to develop Sca AT-domain based vaccines and tools for scrub typhus diagnosis with rapid and cost-effective ways.

Rickettsioses

2020 ◽  
pp. 1230-1251
Author(s):  
Karolina Griffiths ◽  
Carole Eldin ◽  
Didier Raoult ◽  
Philippe Parola
Keyword(s):  
Scrub Typhus ◽  
Spotted Fever ◽  
Intracellular Bacteria ◽  
Spotted Fever Group ◽  
Orientia Tsutsugamushi ◽  
Obligate Intracellular ◽  
Metabolic Characteristics ◽  
The Past ◽  
Life Threatening ◽  
Obligate Intracellular Bacteria

Rickettsioses are mild to life-threatening zoonoses caused by obligate intracellular bacteria of the order Rickettsiales (family Rickettsiaceae). Arthropods, including ticks, fleas, and mites, are implicated as their vectors, reservoirs, or amplifiers. With an increasing number of new pathogens and recognition of new pathogenicity and affected geographical areas over the past few decades, there is a better understanding of the scope and importance of these pathogens, particularly as a paradigm to understanding emerging and remerging infections. The taxonomy has undergone numerous changes, with now three main groups classified as rickettsioses according to morphological, antigenic and metabolic characteristics: (1) Rickettsioses due to the bacteria of the genus Rickettsia, including the spotted fever group, typhus groups (Rickettsiaceae), (2) Ehrlichioses and Anaplasmoses due to bacteria of the Anaplasmataceae and (3) scrub typhus due to Orientia tsutsugamushi.

scholarly journals Type III Secretion System-Dependent Translocation of Ectopically Expressed Yop Effectors into Macrophages by Intracellular Yersinia pseudotuberculosis

2011 ◽  
Vol 79 (11) ◽  
pp. 4322-4331 ◽  
Author(s):  
Yue Zhang ◽  
Galina Romanov ◽  
James B. Bliska
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Bacterial Pathogen ◽  
Type Iii Secretion System ◽  
Inducible Promoter ◽  
Secretion System ◽  
Host Cells ◽  
Content Type ◽  
Type Iii

ABSTRACTYersinia pseudotuberculosisis a Gram-negative bacterial pathogen. Virulence inY. pseudotuberculosisrequires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected withY. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete andY. pseudotuberculosisis internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently whenYersiniais extracellular. However, it is not clear whether the T3SS can be utilized by intracellularY. pseudotuberculosisto translocate Yops. This possibility was investigated here usingY. pseudotuberculosisstrains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viableY. pseudotuberculosisin macrophages. Collectively, our results suggest that the Ysc T3SS ofY. pseudotuberculosiscan function within macrophage phagosomes to translocate Yops into the host cytosol.

scholarly journals Mimicry of the Pathogenic Mycobacterium VacuoleIn VitroElicits the Bacterial Intracellular Phenotype, Including Early-Onset Macrophage Death

2011 ◽  
Vol 79 (6) ◽  
pp. 2412-2422 ◽  
Author(s):  
Julie Early ◽  
Luiz E. Bermudez
Keyword(s):  
Early Onset ◽  
Disease Process ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Content Type ◽  
Time Points ◽  
Stepwise Reduction ◽  
Trace Elemental ◽  
Laboratory Tool

ABSTRACTMycobacterium aviumcomplex (MAC) within macrophages undergoes a phenotype change that allows for more efficient entry into surrounding host cells. We hypothesized that, by developing anin vitrosystem resembling the intravacuolar environment, one could generate insights into the mycobacterial intracellular phenotype. MAC was incubated in “elemental mixtures” that reproduce metal concentrations and pH in the vacuoles at different time points and then used to infect fresh macrophages. Incubation of MAC with the mixture corresponding to the vacuole environment 24 h postinfection infected macrophages at a significantly higher rate than bacteria that were incubated in Middlebrook 7H9 broth. Uptake occurred by macropinocytosis, similar to the uptake of bacteria passed through macrophages. Genes reported to be upregulated in intracellular bacteria, such asMav1365,Mav2409,Mav4487, andMav0996, were upregulated in MAC incubated in the 24-h elemental mixture. Like MAC obtained from macrophages, the vacuoles of bacteria from the 24-h elemental mixture were more likely to contain lysosome-associated membrane protein 1 (LAMP-1). A stepwise reduction scheme of the 24-h elemental mixture indicated that incubation in physiologically relevant concentrations of potassium chloride, calcium chloride, and manganese chloride was sufficient to induce characteristics of the intracellular phenotype. It was demonstrated that bacteria harboring the intracellular phenotype induced early-onset macrophage death more efficiently than bacteria grown in broth. This new trace elemental mixture mimicking the condition of the vacuole at different time points has the potential to become an effective laboratory tool for the study of the MAC andMycobacterium tuberculosisdisease process, increasing the understanding of the interaction with macrophages.

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