The ttsA gene is required for low-calcium-induced type III secretion of Yop proteins and virulence of Yersinia enterocolitica W22703

Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues. At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals. Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins. To identify such genes, we screened transposon insertion mutants of Y. enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein. ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins. ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes. All of these defects were restored by complementation with plasmid-encoded wild-type ttsA. LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal. Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone. To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice. The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain.     

Secretion of hybrid proteins by the Yersinia Yop export system.

After incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of Yersinia spp. release large amounts of a set of plasmid-encoded proteins called Yops. The secretion of these proteins, involved in pathogenicity, occurs via a mechanism that involves neither the removal of a signal sequence nor the recognition of a C-terminal domain. Analysis of deletion mutants allowed the secretion recognition domain to be localized within the 48 N-terminal amino acids of protein YopH, within the 98 N-terminal residues of protein YopE, and within the 76 N-terminal residues of YopQ. Comparison of these regions failed to reveal any sequence similarity, suggesting that the secretion signal of Yop proteins is conformational rather than sequential. Hybrid proteins containing the amino-terminal part of YopH fused to either the alpha-peptide of beta-galactosidase or to alkaline phosphatase deprived of its signal sequence were efficiently secreted to the Yersinia culture medium. This observation opens new prospects in using Yersinia spp. as chimeric-protein producers and as potential live carriers for foreign antigens.     

YscN, the putative energizer of the Yersinia Yop secretion machinery.

Pathogenic yersiniae secrete a set of 11 antihost proteins called Yops. Yop secretion appears as the archetype of the type III secretion pathway. Several components of this machinery are encoded by the virA (lcrA) and virC (lcrC) loci of the 70-kb pYV plasmid. In this paper, we describe yscN, another gene involved in this pathway. It is the first gene of the virB locus. It encodes a 47.8-kDa protein similar to the catalytic subunits of F0F1 and related ATPases, as well as to products of other genes presumed to be involved in a type III secretion pathway. YscN contains the two consensus nucleotide-binding motifs (boxes A and B) described by Walker et al. (J. E. Walker, M. Saraste, M. J. Runswick, and N. J. Gay, EMBO J. 1:945-951, 1982). We engineered a pYV mutant encoding a modified YscN protein lacking box A. This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. We conclude that YscN is a component of the Yop secretion machinery using ATP. We hypothesize that it is either the energizer of this machinery or a part of it.     

PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution

PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37 degrees C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.     

The fliA gene encoding sigma 28 in Yersinia enterocolitica.

Yersinia enterocolitica is an enterobacterium responsible for gastrointestinal syndromes. Its pathogenicity depends on the presence of the 70-kb pYV plasmid, which directs Yop secretion. The Yop secretion machinery, consisting of the YscA-U and LcrD proteins, presents some structural similarity with the flagellum assembly machinery characterized in other bacteria. Flagellum assembly requires sigma 28, an alternative sigma factor. The region upstream of the lcrD gene resembles promoters recognized by sigma 28, suggesting that the similarity between Yop secretion and flagellum assembly could extend to their regulation. The chromosome of Y. enterocolitica also contains pathogenicity determinants such as myfA, which encodes the Myf antigen subunit. The promoter region of myfA also resembles promoters recognized by sigma 28. In an attempt to clarify the role of sigma 28 in the expression of lcrD, myfA, and flagellar genes, we cloned, sequenced, and mutagenized the fliA gene encoding the sigma 28 homolog in Y. enterocolitica. As is the case in other bacteria, fliA was required for motility. However, it was involved neither in fibrilla synthesis nor in Yop secretion. The fliA mutant allowed us to monitor the role of motility in pathogenesis. At least in the mouse model, motility seemed not to be required for Y. enterocolitica pathogenesis.     

TyeA, a protein involved in control of Yop release and in translocation of Yersinia Yop effectors.

Extracellular Yersinia spp. disarm the immune system by injecting the effector Yersinia outer proteins (Yops) into the target cell. Yop secretion is triggered by contact with eukaryotic cells or by Ca2+ chelation. Two proteins, YopN and LcrG, are known to be involved in Yop-secretion control. Here we describe TyeA, a third protein involved in the control of Yop release. Like YopN, TyeA is localized at the bacterial surface. A tyeA knock-out mutant secreted Yops in the presence of Ca2+ and in the absence of eukaryotic cells. Unlike a yopN null mutant, the tyeA mutant was defective for translocation of YopE and YopH, but not YopM, YopO and YopP, into eukaryotic cells. This is the first observation suggesting that Yop effectors can be divided into two sets for delivery into eukaryotic cells. TyeA was found to interact with the translocator YopD and with residues 242-293 of YopN. In contrast with a yopN null mutant, a yopNDelta248-272 mutant was also unable to translocate YopE and YopH. Our results suggest that TyeA forms part of the translocation-control apparatus together with YopD and YopN, and that the interaction of these proteins is required for selective translocation of Yops inside eukaryotic cells.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.     

Assessment of virulence determinants in Yersinia enterocolitica 1A,O:5 strains isolated from chicken carcasses

The virulence potential associated with 42–45 MDa plasmid pYV, of three Yersinia enterocolitica, 1A, O:5, strains isolated previously from chicken carcasses was tested by plasmid extraction, and by four in vitro tests: (1) Auto-agglutination; (2) Calcium dependence for growth at 37 °C; (3) Congo Red and Crystal Violet binding; and (4) Resistance to the bactericidal effect of serum. The in vitro tests were negative in all strains tested, but from strain PP131 it was possible to extract a plasmid which was quite similar in size to the wild type pYV, but heavier and not observed in any other isolated strains. These contradictions between the presence of a plasmid of approximately 42–45 MDa and the in vitro tests negative results may suggest that there is no homology between the PP131 plasmid and pYV. From these results we conclude that the plasmid found in PP131 is not pYV and that the three Y. enterocolitica 1A, O:5, strains are non-pathogenic on pYV classical mechanism basis, which do not exclude the possibility of them becoming infectious by a different mechanism, especially in children or immune-depressed individuals.     

Molecular determinants for virulence in coxsackievirus B1 infection.

Studies demonstrated that a strain derived from an infectious clone of coxsackievirus B1 (CVB1N) (N. Iizuka, H. Yonekawa, and A. Nomoto, J. Virol. 65:4867-4873, 1991) was 3 to 4 log10 less virulent than the myotropic Tucson strain of CVB1 (CVB1T) following intraperitoneal inoculation of newborn mice. Replacement of nucleotides (nt) 69 to 804 from the 5' untranslated region (5' UTR) and 1A coding region of CVB1N or nt 117 to 161 from the 5' UTR with the corresponding part from CVB1T restored greater than 90% of the virulence. Sequencing of the 5' UTR of CVB1T demonstrated areas with a greater similarity to particular echoviruses than to CVB1N, suggesting that recombination events might have occurred, perhaps influencing the virulence phenotype.     

YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion.

Pathogenic yersiniae secrete antihost Yop proteins by a recently discovered secretion pathway which is also encountered in several animal and plant pathogens. The components of the export machinery are encoded by the virA (lcrA), virB (lcrB), and virC (lcrC) loci of the 70-kb pYV plasmid. In the present paper we describe yscU, the last gene of the virB locus. We determined the DNA sequence and mutated the gene on the pYV plasmid. After inactivation of yscU, the mutant strain was unable to secrete Yop proteins. The topology of YscU was investigated by the analysis of YscU-PhoA translational fusions generated by TnphoA transposition. This showed that the 40.3-kDa yscU product contains four transmembrane segments anchoring a large cytoplasmic carboxyl-terminal domain to the inner membrane. YscU is related to Spa40 from Shigella flexneri, to SpaS from Salmonella typhimurium, to FlhB from Bacillus subtilis, and to HrpN from Pseudomonas solanacearum.     

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.     

Dissection of the Yersinia enterocolitica virulence plasmid pYVO8 into an operating unit and virulence gene modules

Abstract Mesophilic Aeromonas hydrophila from serotypes O:11 and O:34 grown in a glucose-rich medium produce a capsule that can be seen under light and electron microscopy. The purified capsular polysaccharide has a composition qualitatively similar for strains O:11 and O:34, but quantitatively different. The capsular polysaccharides were immunogenic in rabbits, and did not cross-react with specific antibodies against either purified lipopolysaccharide from strains O:34 or O:11 or against the S-layer characteristic of strains from serotype O:11.     

Osmoregulation-dependent expression of Yersinia enterocolitica virulence factors.

The effects of hyperosmotic stress on expression of plasmid coded Yop and Yad A proteins--virulence factors of Y.enterocolitica serotype 0:9 were characterized. When cells were shifted to high osmolarity and cultured at 37 degrees C in medium without Ca2+ the production of Yops was inhibited. In contrast, the amount of Yad A protein was unaffected. Addition of glycine betaine to this culture alleviated the effect of high osmolarity. It was also found that hyperosmotic stress causes increased negative supercoiling of DNA in Y. enterocolitica 0:9. Changes in DNA supercoiling coincided with expression of Yop proteins. These results suggest that in high osmolarity the expression of yop genes may be regulated by DNA supercoiling.     

Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.