A deubiquitylase with an unusually high-affinity ubiquitin-binding domain from the scrub typhus pathogen Orientia tsutsugamushi

Berk, Jason M.; Lim, Christopher; Ronau, J.A.; Chaudhuri, Apala; Chen, Hongli; Beckmann, John F.; Loria, J. Patrick; Xiong, Yong; Hochstrasser, Mark

doi:10.1038/s41467-020-15985-4

Cited by 29 publications

(42 citation statements)

References 70 publications

(88 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is weakly active against another Ubl model substrate, NEDD8-AMC [24]. This substrate preference resembles some other bacterial proteins with Ulp1-like domains such as RickCE from Rickettsia bellii and the recently discovered OtDUB from Orientia tsutsugamushi [61,68].…”

Section: Cida-cidb a CI Inducing Protein Pair Containing Deubiquitylmentioning

confidence: 59%

The Biochemistry of Cytoplasmic Incompatibility Caused by Endosymbiotic Bacteria

Chen

Zhang

Hochstrasser

2020

Genes

Self Cite

View full text Add to dashboard Cite

Many species of arthropods carry maternally inherited bacterial endosymbionts that can influence host sexual reproduction to benefit the bacterium. The most well-known of such reproductive parasites is Wolbachia pipientis. Wolbachia are obligate intracellular α-proteobacteria found in nearly half of all arthropod species. This success has been attributed in part to their ability to manipulate host reproduction to favor infected females. Cytoplasmic incompatibility (CI), a phenomenon wherein Wolbachia infection renders males sterile when they mate with uninfected females, but not infected females (the rescue mating), appears to be the most common. CI provides a reproductive advantage to infected females in the presence of a threshold level of infected males. The molecular mechanisms of CI and other reproductive manipulations, such as male killing, parthenogenesis, and feminization, have remained mysterious for many decades. It had been proposed by Werren more than two decades ago that CI is caused by a Wolbachia-mediated sperm modification and that rescue is achieved by a Wolbachia-encoded rescue factor in the infected egg. In the past few years, new research has highlighted a set of syntenic Wolbachia gene pairs encoding CI-inducing factors (Cifs) as the key players for the induction of CI and its rescue. Within each Cif pair, the protein encoded by the upstream gene is denoted A and the downstream gene B. To date, two types of Cifs have been characterized based on the enzymatic activity identified in the B protein of each protein pair; one type encodes a deubiquitylase (thus named CI-inducing deubiquitylase or cid), and a second type encodes a nuclease (named CI-inducing nuclease or cin). The CidA and CinA proteins bind tightly and specifically to their respective CidB and CinB partners. In transgenic Drosophila melanogaster, the expression of either the Cid or Cin protein pair in the male germline induces CI and the expression of the cognate A protein in females is sufficient for rescue. With the identity of the Wolbachia CI induction and rescue factors now known, research in the field has turned to directed studies on the molecular mechanisms of CI, which we review here.

show abstract

Section: Cida-cidb a CI Inducing Protein Pair Containing Deubiquitylmentioning

confidence: 59%

The Biochemistry of Cytoplasmic Incompatibility Caused by Endosymbiotic Bacteria

Chen

Zhang

Hochstrasser

2020

Genes

Self Cite

View full text Add to dashboard Cite

show abstract

“…The periodontal bacterial pathogen, Porphyromonas gingivalis, uses its SerB serine-protease to dephosphorylate NF-κB p65, but it does not act on p105 [58]. O. tsutsugamushi OTT_1962 (OtDUB) was recently structurally and functionally characterized as a deubiquitylase that, when expressed in recombinant form, exhibits a high affinity for and efficiently cleaves polyubiquitin chains [59]. As a role for the newly discovered OtDUB during infection has yet to be determined, it is plausible OtDUB deubiquitylates polyubiquitinated p105.…”

Section: Plos Neglected Tropical Diseasesmentioning

confidence: 99%

Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105

et al. 2021

View full text Add to dashboard Cite

Background Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKβ-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. Principal findings Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen’s ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. Conclusions O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

show abstract

“…The OtDUB N-terminal region contains an active DUB and a high-affinity ubiquitin binding domain (UBD) within the first 259 residues. 11 However, the remainder of the 1369residue protein is devoid of any computationally predicted domains. To examine how the OtDUB might affect eukaryotic cells, we first generated a series of truncations (Fig.…”

Section: The Otdub C-terminal Segment Is Toxic In Yeast and Interacts With Gtpasesmentioning

confidence: 99%

“…The open reading frame (ORF) of OTT_1962 from Orientia tsutsugamushi (Ikeda isolate) was synthesized (by Genscript) with codons optimized for human (primary) and yeast (secondary) expression and inserted into pCDNA3.1(+). 11 The synthesis included a C-terminal Gly-Ser-Gly-1xFlag epitope sequence and 5' BamHI, 3' XhoI restriction sites. This DNA sequence was used as template for all the OtDUB plasmids in this study.…”

Section: Dna and Cloningmentioning

confidence: 99%

Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

Lim

Berk

Blaise

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

17Rho family GTPases regulate an array of cellular processes and are often modulated by 18 pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor 19 (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia 20 tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host 21 interactors, including the Rho-GTPases Rac1 and Cdc42. We discovered a new domain in 22OtDUB with Rac1/Cdc42 GEF activity (OtDUB GEF ), with higher activity toward Rac1 in vitro. 23While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of 24 OtDUB GEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, 25 with a distinct topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF 26 domains. Structure-guided mutational analyses identified residues critical for activity and a novel 27 mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 28 preferentially in cells, and expression of the OtDUB GEF alone alters cell morphology. 29Cumulatively, this work reveals a novel bacterial GEF within the multifunctional OtDUB that 30 co-opts host Rac1 signaling to evoke changes in cytoskeletal structure. 31 motif important for folding and structural integrity) or the SopE family (SopE, SopE2, and 55 BopE). These bacterial effectors share no sequence or structural homology to eukaryotic Rho 56 GEFs, which predominantly belong to the Dbl homology (DH) family of GEFs typically formed 57 by a six-helix bundle with an elongated, kinked "chaise lounge" fold. 8,9 Rather, bacterial effector 58GEFs adopt a characteristic compact V-shaped fold, yet activate the Rho GTPases via the same 59 contact regions in the GTPases that are crucial for nucleotide exchange by DH-family GEFs. 10 60While substantial effort has been exerted in detailing the molecular determinants of bacterial 61 GEF activities and specificities, no bacterial effector GEFs have been identified outside of the 62 WxxxE/SopE-like families. 63Recently, we identified and characterized a putative effector protein, OtDUB, from the 64 obligate intracellular bacterium that causes scrub typhus, Orientia tsutsugamushi. Despite 65 extensive characterization of the OtDUB deubiquitylase (DUB) domain (residues 1-259), the 66 function of the extensive C-terminal region, encompassing more than 1000 amino acids, 67 remained elusive. 11 Here, we report that the OtDUB encodes a novel GEF domain. Using 68 biochemical, structural, and cellular methods, we demonstrate that the OtDUB GEF predominantly 69 activates Rac1 in vitro and in vivo, and that its three-dimensional structure differs drastically in 70 topological and helical arrangement from both the WxxxE and SopE effector GEFs. We further 71 determined the OtDUB GEF :Rac1 complex crystal structure, which reveals that despite its 72 divergence in primary sequence and topology, the OtDUB GEF has convergently evolved the V-73 shaped fold of bacterial GEFs and interacts with ...

show abstract

A deubiquitylase with an unusually high-affinity ubiquitin-binding domain from the scrub typhus pathogen Orientia tsutsugamushi

Cited by 29 publications

References 70 publications

The Biochemistry of Cytoplasmic Incompatibility Caused by Endosymbiotic Bacteria

The Biochemistry of Cytoplasmic Incompatibility Caused by Endosymbiotic Bacteria

Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105

Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

Contact Info

Product

Resources

About