A bacterial type III effector family uses the papain-like hydrolytic activity to arrest the host cell cycle Qing Yaoa,1, Jixin Cuia,b,1, Yongqun Zhua, Guolun Wanga, Liyan Hua, Chengzu Longa, Ran Caoa, Xinqi Liua,2, Niu Huanga, She Chena, Liping Liua, and Feng Shaoa,3 aNational Institute of Biological Sciences, Beijing 102206, China; and bGraduate School of Chinese Academy of Medical Sciences and Beijing Union Medical College, Beijing 100730, China Communicated by Steven L. McKnight, University of Texas Southwestern Medical School, Dallas, TX, January 9, 2009 (received for review October 24, 2008) Pathogenic bacteria deliver effector proteins into host cells rested in the G1 phase, not the G2/M phase (16). Despite the through the type III secretion apparatus to modulate the host strikingly abnormal cell cycle distribution induced by Cif, its bio- function. We identify a family of proteins, homologous to the type chemical activity and the underlying mechanism of Cif function III effector Cif from enteropathogenic Escherichia coli, in pathogens remain completely unknown. including Yersinia, Photorhabdus, and Burkholderia that contain In this work, we identify a family of known and putative type III functional type III secretion systems. Like Cif, this family of proteins effectors from Yersinia, Photorhabdus, and Burkholderia showing is capable of arresting the host cell cycle at G2/M. Structure of one sequence homology to Cif, and we demonstrate that they all harbor of the family members, Cif homolog in Burkholderia pseudomallei a sufficient and potent G2/M arrest activity. The crystal structure of (CHBP), reveals a papain-like fold and a conserved Cys-His-Gln Cif homolog in Burkholderia pseudomallei (CHBP) reveals a papa- catalytic triad despite the lack of primary sequence identity. For in-like fold with a well-conserved Cys-His-Gln catalytic triad. The CHBP and Cif, only the putative catalytic Cys is susceptible to Cys is susceptible to covalent modification by E-64, a papain covalent modification by E-64, a specific inhibitor of papain-like family-specific inhibitor. A characteristic negatively charged pocket cysteine proteases. Unlike papain-like enzymes where the S2 site that could best fit an arginine likely serves as a distinct substrate- BIOCHEMISTRY is the major determinant of cleavage-site specificity, CHBP has a binding subsite. Mutations disrupting the catalytic triad or the characteristic negatively charged pocket occupying surface areas negatively charged pocket abolish cell cycle arrest function of the corresponding to the S1/S1؅ site in papain-like proteases. The Cif family. Our results demonstrate that bacterial pathogens have negative charge is provided by a conserved aspartate, and the evolved a unique papain-like hydrolytic activity to target and pocket best fits an arginine, as revealed by molecular docking paralyze the eukaryotic cell cycle machinery. analysis. Mutation analysis establishes the essential role of the catalytic triad and the negatively charged pocket in inducing cell Results cycle arrest in host cells. Our results demonstrate that bacterial Identification of a Type III Effector Family of Cell Cycle Modulators. pathogens have evolved a unique papain-like hydrolytic activity to EPEC Cif contains 282 aa with no domains recognized by SMART block the normal host cell cycle progression. (http://smart.embl-heidelberg.de/) or Pfam (http://pfam.janeli- a.org/). Extensive sequence analysis employing PSI-BLAST itera- Burkholderia ͉ crystal structure ͉ cyclomodulin ͉ type III secretion ͉ EPEC tions (17) revealed no homologous proteins with known functions and instead led to identification of three new homologous bacterial ram-negative bacterial pathogens, such as Shigella, Salmonella, proteins. They are named CHYP (Cif homolog in Yersinia pseudo- GYesinia, and enteropathogenic Escherichia coli (EPEC), em- tuberculosis), CHPL (Cif homolog in Photorhabdus luminescens), ploy a specialized type III secretion system (TTSS) to inject effector and CHBP. As shown in Fig. 1A, CHYP has the highest degree of proteins into host cells (1). These effectors function to interfere sequence homology to Cif (Ϸ54% identity and Ϸ70% similarity); with various host signal transduction pathways, most frequently the CHPL and CHBP are distantly related and only exhibit Ϸ20% innate immune system (2), for the benefits of pathogen survival and sequence identity to Cif. The sequence conservation among the systemic infection (3, 4). Elucidating biochemical functions of type entire Cif family lies mainly in the middle and C-terminal regions, III effectors, particularly those conserved among different patho- and their more diverse N-terminal sequences agree with the gens, has greatly promoted our understanding of novel yet common prediction that they are all TTSS effectors (see below). mechanisms of bacterial pathogenesis (5–7). Similarly to Cif-harboring EPEC, Y. pseudotuberculosis causes In addition to the innate immune pathway, recent studies suggest gastroenteritis in rodents, and CHYP can only be identified in Y. that the host cell cycle machinery represents an emerging target of pseudotuberculosis YPIII strain. P. luminescens is a symbiont of bacterial toxins, including some type III effectors (8–11). The nematodes, and the infection kills a broad spectrum of insects. The Shigella type III effector IpaB binds directly to Mad2L2 to release its inhibitory effect on the anaphase-promoting complex/ cyclosome, thereby causing a delayed mitotic progression or cell Author contributions: J.C. and F.S. designed research; Q.Y., J.C., Y.Z., L.H., C.L., R.C., X.L., and N.H. performed research; G.W., S.C., and L.L. contributed new reagents/analytic tools; Q.Y., cycle arrest at the G2/M phase (9). This serves as an important J.C., and F.S. analyzed data; and Q.Y., J.C., and F.S. wrote the paper. strategy for Shigella to colonize the gut epithelium efficiently. The authors declare no conflict of interest. Another bacterial toxin called cytolethal distending toxin (CDT) Data deposition: The atomic coordinates and structure factors for CHBP have been depos- uses a DNase I-like activity to trigger the DNA damage checkpoint ited in the Protein Data Bank, www.pdb.org (PDB ID codes 3EIR and 3EIT). and block the host cell cycle at the G2/M transition (12, 13). 1Q.Y. and J.C. contributed equally to this work. Similarly, a G /M arrest phenotype is observed upon infection of 2 2Present address: College of Life Science, Nankai University, Tianjin 300071, China. epithelial cells by certain strains of EPEC because of the activity of 3To whom correspondence should be addressed at: National Institute of Biological Sciences, a newly identified type III effector called Cif (cycle-inhibiting 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China. E-mail: factor) (14). In contrast to CDT, Cif does not activate the DNA [email protected]. damage checkpoint (15). Interestingly, a recent study shows that This article contains supporting information online at www.pnas.org/cgi/content/full/ post-G2 cells infected with Cif-expressing EPEC are instead ar- 0900212106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900212106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Identification of the Cif family of cell cycle modulators. (A) Multiple sequence alignment of the Cif family of effectors. The Cif fam- ily was identified by BLAST searches using the sequence of Cif from EPEC. Alignment was generated by using the ClustalW program with manual adjustments based on pre- dicted secondary structures. The protein names are indicated at the beginning of the alignment: CHYP, from Y. pseudotuberculosis YPIII (ORF name, YPK࿝1971); CHPL, from P. luminescens TTO1 (ORF name, plu2515); CHBP, from B. pseudoma- llei K96243 (ORF name, BPSS1385). Secondary structure elements de- termined from the CHBP crystal (see Fig. 2) are indicated on top of the sequence with ␣-helix in green and ␤-strand in pink. Identical residues are highlighted in black, and similar residues are in gray. Functionally important residues (see Figs. 3B and 5) are colored in yellow or red. (B) Effects of the Cif family members on eukaryotic cell cycle progression. Shown is the flow cytometry analy- sis of DNA contents of EGFP-positive 293T cells transfected with indi- cated EGFP-tagged constructs. gene encoding CHPL is present in the genome of P. luminescens karyotic cell cycle progression. Similar to that observed with EPEC TTO1 strain, which contains an unprecedented large number of infection, ectopically expressed EGFP-Cif induced marked accu- potential virulence genes (18) and a TTSS cluster essential for mulation of 293T cells with 4C DNA content (Fig. 1B). Cells colonization in insects (19). B. pseudomallei, classified as a category transfected with EGFP-CHBP or EGFP-CHPL were also arrested B agent by the US Centers for Disease Control and Prevention, is at the G2/M transition (Fig. 1B). These results suggest that CHBP a potential bioterrorism agent. Infection with the bacteria causes from B. pseudomallei and CHPL from P. luminescens are functional melioidosis, a severe human disease with a high mortality (20–50%) homologs of Cif from EPEC, and the entire Cif family effectors and endemic to areas of Southeast Asia and Northern Australia likely employ a similar biochemical mechanism in blocking the host (20). Notably, sequenced genomes of Ͼ10 different B. pseudomallei cell cycle progression. clinical isolates all contain the CHBP gene, which is absent from genomes of closely related Burkholderia thailandensis and Burk- Overall Structure of CHBP. To understand the nature of the Cif family holderia mallei that usually do not cause human melioidosis. Com- of cell cycle modulators and gain mechanistic insights into their parative genomic analysis of B. pseudomallei and other related functions, we purified recombinant CHBP-N48 (truncation of the bacteria reveals numerous horizontally acquired genomic islands N-terminal 47 residues) and determined the 2.1-Å crystal structure and three TTSSs (21). Among them, TTSS3 shares homology with by Se-SAD (single-wavelength anomalous diffraction) (Fig.