<i>Ehrlichia</i>
            type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation

Yan, Qi; Lin, Mingqun; Huang, Weiyan; Teymournejad, Omid; Johnson, Jennifer M.; Hays, Franklin A.; Liang, Zong-Qi; Li, Guangpu; Rikihisa, Yasuko

doi:10.1073/pnas.1806904115

Cited by 44 publications

(61 citation statements)

References 68 publications

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“…Listeria monocytogenes [81] and Tropheryma whipplei [82] block Rab5 activation, thus arresting phagosome maturation. The Ehrlichia chafeensis T4SS effector Etf-2 binds active Rab5 on bead phagosomes and blocks subsequent recruitment of other proteins, effectively inhibiting endosome maturation [59]. Therefore, it is possible that the C. burnetii T4BSS dysregulates Rab5 to Rab7 conversion as a mechanism to inhibit endosome maturation.…”

Section: Discussionmentioning

confidence: 99%

“…Rab5 regulates fusion between newly formed vesicles carrying cargo and pre-existing early endosomes [57], with Rab5 being replaced by Rab7 as early endosomes mature to late endosomes [58]. Because Rab5 to Rab7 conversion is a critical step during endosomal maturation, we tested whether C. burnetii T4BSS affects Rab5 to Rab7 conversion using co-localization with fluorescent beads [59]. Due to the reduced phagocytic capacity of HeLa cells, we used MH-S macrophages.…”

Section: Burnetii T4bss Alters Latex Bead Phagosome Maturationmentioning

confidence: 99%

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Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

et al. 2019

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Upon host cell infection, the obligate intracellular bacterium Coxiella burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring endosomal and lysosomal markers, as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, inducing CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates pH of the mature CCV. Because heterotypic fusion with host endosomes/ lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii. In WT-infected cells, we observed a significant decrease in proteolytically active, LAMP1-positive endolysosomal vesicles, compared to mock or ΔdotA-infected cells. Using a ratiometric assay to measure endosomal pH, we determined that the average pH of terminal endosomes in WT-infected cells was pH~5.8, compared to pH~4.75 in mock and ΔdotA-infected cells. While endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells. Finally, increasing lysosomal biogenesis by overexpressing the transcription factor EB resulted in smaller, more proteolytically active CCVs and a significant decrease in C. burnetii growth, indicating host lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii inhibits endosomal maturation to reduce the number of proteolytically active lysosomes available for heterotypic fusion with the CCV, possibly as a mechanism to regulate CCV pH.The obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. Here, we discovered that C. burnetii blocks endolysosomal maturation and acidification during host cell infection, resulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the host cells inhibited C. burnetii growth. Together, our study suggests that C. burnetii regulates vacuole acidity and blocks endosomal maturation in order to produce a permissive intracellular niche.Coxiella manipulates endosom...

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Section: Discussionmentioning

confidence: 99%

Section: Burnetii T4bss Alters Latex Bead Phagosome Maturationmentioning

confidence: 99%

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

et al. 2019

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“…Rab5 regulates fusion of newly formed vesicles carrying cargo with pre-existing early endosomes [42], followed by maturation to late endosomes with Rab5 being replaced by Rab7 [43]. Because Rab5 to Rab7 conversion is critical for endosomal maturation, we tested whether C. burnetii T4BSS affects Rab5 to Rab7 conversion using co-localization with fluorescent beads [44]. Due to the reduced phagocytic capacity of HeLa cells, we used murine alveolar macrophages (MH-S).…”

Section: Resultsmentioning

confidence: 99%

“…Listeria monocytogenes [64] and Tropheryma whipplei [65] block Rab5 activation, thus arresting phagosome maturation. The Ehrlichia chafeensis T4SS effector Etf-2 binds active Rab5 on bead phagosomes and blocks subsequent recruitment of other proteins, effectively inhibiting endosome maturation [44]. Therefore, it is possible that the C. burnetii T4BSS dysregulates Rab5 to Rab7 conversion as a potential mechanism to inhibit endosome maturation.…”

Section: Discussionmentioning

confidence: 99%

Coxiella burnetiiType 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Samanta

Clemente

Gilk

2019

Preprint

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29Upon host cell infection, the obligate intracellular bacterium C. burnetii resides and multiplies 30 within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the 31 endosomal maturation pathway into a phagolysosome, acquiring lysosomal markers as well as 32 acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, 33 heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion 34 and subsequent bacterial replication in the mature CCV. Initial CCV acidification is required to 35 activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes 36 effector proteins required for CCV maturation. However, we recently found that the mature CCV 37 is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, CCV acidification to pH~4.8 causes C. 38 burnetii lysis, suggesting C. burnetii actively regulates CCV pH. Because heterotypic fusion with 39 host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in 40 cells infected with wildtype (WT) or T4BSS mutant (dotA) C. burnetii. We observed significantly 41 fewer LAMP1-positive lysosomes, along with less acidic "mature" endosomes (pH~5.8), in WT-42 infected cells, compared to mock or dotA-infected cells. Further, while endosomes progressively 43 acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and dotA-44 infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells, indicating that the 45 C. burnetii T4BSS inhibits endosomal maturation. Finally, increasing the number of acidic 46 lysosomes by overexpressing the transcription factor EB inhibited C. burnetii growth, indicating 47 lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii regulates CCV 48 pH, possibly by reducing the number of host lysosomes available for heterotypic fusion. 49 50 51 3 52 Author summary 53 54 The obligate intracellular bacterium Coxiella burnetii causes human Q fever, which manifests as 55 a flu-like illness but can develop into a life-threatening and difficult to treat endocarditis. C. burnetii, 56in contrast to many other intracellular bacteria, thrives within a lysosome-like vacuole in host cells. 57However, we previously found that the C. burnetii vacuole is not as acidic as lysosomes and 58 increased acidification kills the bacteria, suggesting that C. burnetii regulates the pH of its vacuole. 59Here, we discovered that C. burnetii blocks endosomal maturation and acidification during hostresulting in fewer lysosomes in the host cell. Moreover, increasing lysosomes in the 61 host cells blocked C. burnetii growth. Together, our study suggests that C. burnetii regulates 62 vacuole acidity and blocks endosomal acidification in order to produce a permissive intracellular 63 niche. 64 65 79 infection, phagosome expansion, presumably through fusion with the endocytic pathway, gives 80 rise to a large acidic phagolysosomal-like compartment termed the Coxiella-containing vacuole 81 (CCV) [...

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“…No known pathogen-associated molecular patterns (PAMPs) including endotoxin, peptidoglycan, flagella, common pili, or exotoxin has been detected in Ehrlichia spp., yet they induce acute and/or chronic inflammatory cytokines production in MyD88-dependent, but Toll-like receptors (TLR)-independent manner (Koh et al, 2010;Miura et al, 2011;Rikihisa, 2015). Several virulence factors of E. chaffeensis, which are required for bacterial entry, survival and proliferation of macrophages, have been demonstrated, including ehrlichial invasin that binds host cell receptor and triggers its entry into host cells, three type IV secretion system effectors, three pairs of two-component regulatory system, and outer membrane porins for nutrient acquisition (Kumagai et al, 2006(Kumagai et al, , 2008Mohan Kumar et al, 2013;Rikihisa, 2015Rikihisa, , 2017Lin et al, 2016;Sharma et al, 2017;Teymournejad et al, 2017;Yan et al, 2018). However, whether Ehrlichia spp.…”

Section: Introductionmentioning

confidence: 99%

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

Bekebrede

Lin

Teymournejad

et al. 2020

Front. Cell. Infect. Microbiol.

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Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multicopy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1β and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.Keywords: Ehrlichia HF strain, Himar1 transposon mutagenesis, mouse virulence, inflammatory cytokines, staphylococcal superantigen-like domain, obligatory intracellular bacteria IMPORTANCE Ehrlichiosis, a sometimes deadly febrile disease in man and animal, is caused by infection with Gram-negative obligatory intracellular bacteria, Ehrlichia. Ehrlichia species lack typical pathogen-associate molecular patterns (PAMPs), such as lipopolysaccharide, peptidoglycan, pili, and flagella, yet they induce acute and/or chronic inflammatory cytokines in infected animals.Bekebrede et al. In vivo Virulence Genes of EhrlichiaStudies to identify virulence factors and PAMPs of Ehrlichia species are hampered by the limitation to applicable genetic tools and small laboratory animal models. Mouse infection with Ehrlichia HF strain provides an excellent model for fatal human ehrlichiosis, as the HF strain is highly virulent in laboratory mice. However, due to inability to culture the HF stain, the use of this model has been limited. As we have succeeded in culturing and who...

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Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation

Cited by 44 publications

References 68 publications

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Coxiella burnetiiType 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

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