Development of TEM-1 β-lactamase based protein translocation assay for identification of Anaplasma phagocytophilum type IV secretion system effector proteins

Zhu, Jiafeng; He, Meiling; Xu, Wenting; Li, Yuanyuan; Huang, Rui; Wu, Shuyan; Niu, Hua

doi:10.1038/s41598-019-40682-8

Cited by 9 publications

(5 citation statements)

References 40 publications

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“…We found that nucleolin was pulled down only from AFAP-SF-expressing cells but not from SF tag-expressing cells and APH0215-SF-expressing cells, indicating that the interaction between AFAP and nucleolin was specific ( Figure S1 ). Of note, APH0215 is an A. phagocytophilum protein [ 29 ]. APH0215-SF, which harbors SBP-3×FLAG, was constructed (Yu et al, unpublished data) and used here as a negative control.…”

Section: Resultsmentioning

confidence: 99%

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP

Tang

Zhang

Jiang

et al. 2023

JPM

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Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular Gram-negative bacterium. During infection, A. phagocytophilum enhances the adhesion of neutrophils to the infected endothelial cells. However, the bacterial factors contributing to this phenomenon remain unknown. In this study, we characterized a type IV secretion system substrate of A. phagocytophilum, AFAP (an actin filament-associated Anaplasma phagocytophilum protein) and found that it dynamically changed its pattern and subcellular location in cells and enhanced cell adhesion. Tandem affinity purification combined with mass spectrometry identified host nucleolin as an AFAP-interacting protein. Further study showed the disruption of nucleolin by RNA interference, and the treatment of a nucleolin-binding DNA aptamer AS1411 attenuated AFAP-mediated cell adhesion, indicating that AFAP enhanced cell adhesion in a nucleolin-dependent manner. The characterization of cell adhesion-enhancing AFAP and the identification of host nucleolin as its interaction partner may help understand the mechanism underlying A. phagocytophilum-promoting cell adhesion, facilitating the elucidation of HGA pathogenesis.

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Section: Resultsmentioning

confidence: 99%

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP

Tang

Zhang

Jiang

et al. 2023

JPM

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“…In addition, the transcription regulator tr1 (HGE1_05312), shown to be upregulated 9.1-fold in sample 3 (2 h with ISE6) relative to sample 1 (2 h with HL-60; Table S6A), was the most upregulated gene in sample 5 (9.9fold). Also upregulated in sample 5, and in sample 3 relative to sample 1, was HGE1_00935, which encodes an effector protein that interacts with the trans-Golgi network (27). Through contact with ISE6 cells, the HL-60-grown bacteria (sample 5) shifted their transcription, and early ISE6-specific effectors and membrane proteins were upregulated.…”

Section: Transcription Associated With Host Cell Binding/entrymentioning

confidence: 99%

“…Genes that were upregulated 2-to 3-fold in sample 1 included: HGE1_03697, encoding Ats-1, which is imported to mitochondria, inhibits apoptosis and induces autophagosome formation (26); HGE1_01090, encoding Asp14, which is surface-expressed and critical for infection (31); and HGE1_03902, encoding AipA, an invasin necessary for infection of mammalian cells (25). HGE1 genes upregulated in sample 3 (2 h with ISE6 cells) included the p44ES transcription regulator, tr1 [HGE1_05312; (19)], which was the second-most elevated (9.1-fold, Figure S5), and likely drives expression of p44 paralogs specifically expressed in ISE6 cells, i.e., HGE1_05312 ( Figure S5), and several others at later times; HGE1_00935 (upregulated 9.9-fold in sample 5) encoding an effector that interacts with the trans-Golgi network (27); and 39 hypothetical genes, 19 of which were predicted to be secreted or localized to the bacterial membranes or periplasm.…”

Section: Transcriptional Differences Between Bacteria Binding/enterinmentioning

confidence: 99%

Global Transcription Profiles of Anaplasma phagocytophilum at Key Stages of Infection in Tick and Human Cell Lines and Granulocytes

et al. 2020

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The incidence of human diseases caused by tick-borne pathogens is increasing but little is known about the molecular interactions between the agents and their vectors and hosts. Anaplasma phagocytophilum (Ap) is an obligate intracellular, tick-borne bacterium that causes granulocytic anaplasmosis in humans, dogs, sheep, and horses. In mammals, neutrophil granulocytes are a primary target of infection, and in ticks, Ap has been found in gut and salivary gland cells. To identify bacterial genes that enable Ap to invade and proliferate in human and tick cells, labeled mRNA from Ap bound to or replicating within human and tick cells (lines HL-60 and ISE6), and replicating in primary human granulocytes ex vivo, was hybridized to a custom tiling microarray containing probes representing the entire Ap genome. Probe signal values plotted over a map of the Ap genome revealed antisense transcripts and unannotated genes. Comparisons of transcript levels from each annotated gene between test conditions (e.g., Ap replicating in HL-60 vs. ISE6) identified those that were differentially transcribed, thereby highlighting genes associated with each condition. Bacteria replicating in HL-60 cells upregulated 122 genes compared to those in ISE6, including numerous p44 paralogs, five HGE-14 paralogs, and 32 hypothetical protein genes, of which 47% were predicted to be secreted or localized to the membrane. By comparison, 60% of genes upregulated in ISE6 encoded hypothetical proteins, 60% of which were predicted to be secreted or membrane associated. In granulocytes, Ap upregulated 120 genes compared to HL-60, 33% of them hypothetical and 43% of those predicted to encode secreted or membrane associated proteins. HL-60-grown bacteria binding to HL-60 cells barely responded transcriptionally, while ISE6-grown bacteria binding to ISE6 cells upregulated 48 genes. HL-60-grown bacteria, when incubated with ISE6 cells, upregulated the same genes that were upregulated by ISE6-grown bacteria exposed to uninfected ISE6. Hypothetical genes (constituting about 29% of Ap genes) played a disproportionate role in most infection scenarios, and particular sets of them were consistently upregulated in bacteria binding/entering both ISE6 and HL-60 cells. This suggested that the encoded proteins played central roles in establishing infection in ticks and humans.

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“…The rvh T4SS is odd in its design 41,42 , with specialized duplications of some components hypothesized to autoregulate effector secretion 43,44 . Effectors have been characterized for species of Ehrlichia [45][46][47][48] , Anaplasma [49][50][51][52][53][54] , and Rickettsia [55][56][57] . Schön et al proposed that species of Mitibacteraceae and Athabascaceae utilize the rvh T4SS for killing congener microbes 28 , provided that these genomes harbor candidate rvh effectors with characteristics similar to effectors in other T4SS and T6SS killing machines 58,59 .…”

Section: Introductionmentioning

confidence: 99%

Metagenome diversity illuminates origins of pathogen effectors

Verhoeve

Lehman

Driscoll

et al. 2023

Preprint

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Recent metagenome assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. Discovery of basal lineages (Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles reveals an evolutionary timepoint for the transition to host dependency, which occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system (T4SS) and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for derived rickettsial pathogens. MAG analysis also substantially increased diversity for genus Rickettsia and delineated a basal lineage (Tisiphia) that stands to inform on the rise of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages indicates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, illuminating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role shaping the rvh effector landscape, as evinced by the discover of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can provide incredible insight on the origins of pathogen effectors and how their architectural modifications become tailored to eukaryotic host cell biology.

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Development of TEM-1 β-lactamase based protein translocation assay for identification of Anaplasma phagocytophilum type IV secretion system effector proteins

Cited by 9 publications

References 40 publications

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP

Enhancement of Cell Adhesion by Anaplasma phagocytophilum Nucleolin-Interacting Protein AFAP

Global Transcription Profiles of Anaplasma phagocytophilum at Key Stages of Infection in Tick and Human Cell Lines and Granulocytes

Metagenome diversity illuminates origins of pathogen effectors

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