INFECTION AND IMMUNITY, Jan. 1993, p. 71-80 Vol. 61, No. 1 0019-9567/93/010071-10$02.00/0 Copyright © 1993, American Society for Microbiology YopB and YopD Constitute a Novel Class of Yersinia Yop Proteins SEBASTIAN HAKANSSON,1 THOMAS BERGMAN,1 JEAN-CLAUDE VANOOTEGHEM, 2 GUY CORNELIS,2 AND HANS WOLF-WATZ1* Department of Cell and Molecular Biology, University of Umed, S-901 87 Umed, Sweden,' and Microbial Pathogenesis Unit, Intemnational Institute of Cellular and Molecular Pathology and Faculte6 de Medecine, Universite Catholique de Louvain, B-1200 Brussels, Belgium2 Received 21 May 1992/Accepted 21 October 1992 Virulent Yersinia species harbor a common plasmid that encodes essential virulence determinants (Yersinia outer proteins [Yops]), which are regulated by the extracellular stimuli Ca2" and temperature. The V-antigen-encoding operon has been shown to be involved in the Ca2 -regulated negative pathway. The genetic organization of the V-antigen operon and the sequence of the krGVH genes were recently presented. The V-antigen operon was shown to be a polycistronic operon having the gene order kcrGVH-yopBD (T. Bergman, S. Hakansson, A. Forsberg, L. Norlander, A. Maceliaro, A. Backman, I. Bolin, and H. Wolf-Watz, J. Bacteriol. 173:1607-1616, 1991; S. B. Price, K. Y. Leung, S. S. Barve, and S. C. Straley, J. Bacteriol. 171:5646-5653, 1989). We present here the sequence of the distal part of the V-antigen operons of Yersinia pseudotuberculosis and Yersinia enterocolitica. The sequence information encompasses theyopB andyopD genes and a downstream region in both species. We conclude that the V-antigen operon ends with theyopD gene. This conclusion is strengthened by the observation of an insertion-like element downstream of the yopD gene. The translational start codons of YopB and YopD have been identified by N-terminal amino acid sequencing. By computer analysis, the yopB and yopD gene products were found to be possible transmembrane proteins, and YopD was shown to contain an amphipathic a-helix in its carboxy terminus. These findings contrast with the general globular pattern observed for other Yops. Homology between Yersinia LcrH and Shigellalzexneri IppI and between Yersinia YopB and S. flexneri IpaB was found, suggesting conservation of this locus between these two genera. YopB was also found to have a moderate level of homology, especially within the hydrophobic regions, to members of the RTX protein family of alpha-hemolysins and leukotoxins, indicating that YopB might exhibit a similar function. The genus Yersinia consists of 10 species, of which 3 in Ca2"-depleted conditions, the cells are restricted for (Yersinia pestis, Y pseudotuberculosis, and Y enterocoli- growth after approximately two generations (68). Concomi- tica) are virulent. Common to all three virulent species is a tantly with the low-calcium response, the virulence plasmid- plasmid of approximately 70 kb that is essential for virulence encoded Yop proteins are expressed and secreted in large (23-25, 46, 69). This virulence plasmid encodes a number of amounts into the culture supernatant. If the pathogen is secreted proteins called Yersinia outer proteins (Yops) (7, grown at 26°C or in the presence of 2.5 mM Ca2+, synthesis 14, 61). The yop genes are scattered around the virulence of Yop proteins is repressed. plasmid and have been shown to be contained in different The expression of theyop genes is under the control of the operons (8, 15, 21, 34, 40, 42, 61). A number of these Yops extracellular stimuli temperature and Ca2+ (8, 13, 14, 20, 21, have been shown to be virulence determinants, since specific 61). This regulation of expression is controlled by two yop mutants generally are avirulent (8, 21, 42, 61). The different regulatory loops, one positive and one negative (3, YopH and YopE proteins are involved in resistance of 8, 13, 21, 48). The positive loop involves one activator, VirF yersiniae to phagocytosis by macrophages. Therefore, these (LcrF), which exhibits sequence homology to AraC of two proteins are implicated in the obstruction of the primary Escherichia coli (13, 14) and interacts with the yop promot- host defense.(51, 52). The YopE protein has been shown to ers (32). When the cells are shifted from 26 to 37°C, the level be a cytotoxic effector and to be involved in resistance to of VirF increases, even in the presence of 2.5 mM Ca2 , phagocytosis (52). YopH was found to exhibit a protein suggesting that the positive loop involves only temperature tyrosine phosphatase activity, suggesting that YopH inter- as a stimulus (13). The Ca2+ concentration of the growth feres with regulatory or signal-transducing pathways of medium regulates the level of Yop expression at 37°C. This target cells (5, 26). The YopM protein exhibits homology to effect is likely mediated through the expression of a tran- the a-chain of human platelet glycoprotein Ib, GPIba, and scriptional repressor that interacts with the yop promoters was shown to inhibit human platelet aggregation (34, 35). (3, 8, 21, 51). At a Ca2+ concentration of at least 2.5 mM, the The virulence plasmid has also been shown to be associ- putative repressor concentration is high, resulting in repres- ated with the unique low-Ca2" response of pathogenic sion of theyop genes. Growth in a calcium-depleted medium yersiniae (8, 14, 15, 20-22, 25, 30, 45, 46, 61). This low-Ca2+ results in a low concentration of an active transcriptional response is manifested by the requirement of at least 2.5 mM repressor and, accordingly, derepression of the yop genes. Ca"+ for prolonged growth at 37°C. Upon incubation at 37°C The complex negative regulatory loop involves at least five different loci (20). One of these loci is the V-antigen- containing operon (3, 42, 61). Mutations in this operon give * Corresponding author. the bacteria a temperature-sensitive phenotype (i.e., the 71 72 HAKANSSON ET AL. INFECT. IMMUN. mutant shows a reduced plating frequency at 37°C irrespec- yopD genes and downstream sequences of Y pseudotuber- tive of Ca2+ concentration), indicating the importance of this culosis were subcloned into the pBluescript vector (Strata- operon in the negative control of Yop expression (3, 14, 21, gene, La Jolla, Calif.). Polymerase chain reaction (PCR) 42, 49). This operon has previously been shown to contain at fragments derived from the Y enterocolitica virulence plas- least five genes: lcrGVI-yopBD (3, 42, 48). A regulatory role mid pYVe227, which contains the yopB andyopD genes and for both the LcrV and LcrH proteins has been demonstrated a downstream region, were cloned into either the pBC18 (3, 47, 49). The LcrV protein is suggested to be involved in vector (10) or the pBluescript vector. The subclonings of the secretory mechanisms, while the LcrH protein is proposed PCR fragments from Y enterocolitica were performed at to be a negative control element of yersiniae (3, 48, 49). two separate times. The DNA sequences of these parallel Polar mutations of the yopB and yopD genes gave rise to subclones of Y enterocolitica and the subclones of Y temperature-sensitive mutants (3, 20). This was probably pseudotuberculosis were determined by the dideoxy-chain due to a destabilization of the transcript which negatively termination method of Sanger et al. (54) with [a-35S]dATP influences the expression of LcrH (3). Thus, in order to (Amersham Corp., Little Chalfont, United Kingdom) and study the function of YopB and YopD, it was necessary to modified T7 DNA polymerase (Sequenase; United States obtain the sequence information of this distal part of the Biochemical Corp., Cleveland, Ohio). V-antigen operon so that specific mutants could be con- DNA and protein sequences were analyzed by the GCG structed. (Genetics Computer Group sequence analysis software So far, little is known about YopB and YopD. Rosqvist package [University of Wisconsin, Madison]) and PC Gene has shown by immunostaining that the yopB gene product (IntelliGenetics, Inc., Mountain View, Calif.) computer pro- binds to the same cytoskeleton structure in the target cell as grams. The FASTA and TFASTA homology searches of the cytotoxin YopE. This finding could indicate a role in GCG were performed with the algorithms of Lipman and virulence for the YopB protein (50). YopD, on the other Pearson (36) and Pearson and Lipman (44). hand, has been implicated in the cytotoxic response or in the The mean a-helical hydrophobic moment and the mean process of translocation of the YopE cytotoxin across the hydrophobicity were calculated by the procedure of Eisen- target cell membrane, as deduced by the use of microinjec- berg et al. (16, 17). A segment of 11 amino acids was moved tion techniques (53). through the protein sequence, and the mean hydrophobicity In this study, we demonstrated by sequence analysis that moments for each segment were calculated. The mean the V-antigen-containing operon ends with the yopD gene hydrophobicity was plotted against the mean a-helical hy- and that the YopB and YopD proteins are of the same size drophobic moment for all possible segments. These two and are highly conserved in Y pseudotuberculosis and Y. parameters were also plotted as a function of the midpoint of enterocolitica. We showed that YopB and YopD are possi- the amino acid segment along the sequence. In most calcu- ble transmembrane proteins and that YopD contains an lations, we used the normalized consensus hydrophobic amphipathic domain in its carboxy terminus. We also scale of Eisenberg et al., which is especially suitable for showed that LcrH and YopB exhibit homology with IppI and membrane-related proteins (16, 17). IpaB of shigellae, respectively (2, 56, 65, 67), and that YopB, Analysis of Yop expression. Yersinia strains were grown at in addition, exhibits moderate homology with the RTX 26°C to an optical density of 0.1 at 550 nm.