Sli Gene Research Articles

Reduced virulence is an important characteristic of biofilm infection of Streptococcus suis

Streptococcus suis 2 (SS2) is a zoonotic pathogen that can participate in biofilm formation to survive in hostile environments. In this study, virulent SS2 strains HA9801 and ZY05719 displayed increased biofilm formation compared with SS2 avirulent strain T15. In addition, a 58% reduction in adherence to HEp-2 cells was observed for HA9801 biofilm cells, compared with HA9801 planktonic cells. The 50% lethal dose (LD(50) ) of biofilm cells was 40-fold greater than that of planktonic cells. Quantification of expression levels of known virulence genes by real-time PCR revealed that the transcription levels of the gdh, cps2 and mrp genes in biofilm cells were downregulated, while the sly and gapdh genes were upregulated. HA9801 biofilm and planktonic vaccines provided 60% and 46% protection, respectively, when challenged with 50 times the LD(50) of the HA9801 strain. These results suggest a possible connection between virulence and the ability of biofilm formation; cell adhesion, transcription levels and virulence properties are different between biofilm cells and planktonic cells. Furthermore, this work offers a novel insight into bacterium infection mechanisms, which suggests that a virulent strain may be able to decrease its virulence by forming a biofilm so that it can achieve persistent infection in vivo.

Sly Represses Gene Expression in Sex Chromosomes

The nuclei of eukaryotic cells can be thought of as a library containing the molecular “recipes,” or genes, for constructing the proteins and ribonucleic acid molecules used by a cell. Within the library, genes are grouped together into “books,” known as chromosomes, which consist of linear strands of DNA wrapped around specialized DNA-interacting proteins. Human body cells have 46 chromosomes. Of these, there are two copies of each of 22 unique chromosomes (one inherited from the mother and one from the father). The remaining two chromosomes are the sex chromosomes—two X chromosomes in a female and an X with a Y chromosome in a male. The Y chromosome is responsible for triggering the development of male physical characteristics. However, the functions of most Y-chromosomal genes are unknown. In this issue of PLoS Biology, Julie Cocquet, Paul Burgoyne, and colleagues provide new insights into the role of the mouse Y-chromosome gene Sly. Until now, Sly's role in cells has been a mystery, because unlike most genes, which have just one copy, Sly is present in multiple (over 100) copies. The conventional approach to studying a gene's function—knocking it out—involves replacing a normal gene with a disrupted version containing a selectable marker. This approach is unworkable with Sly, and other multicopy genes, because there are not enough selectable markers or man hours available to engineer that many disrupted copies. Therefore, the authors took an alternative approach: they designed transgenes to generate short hairpin RNAs (shRNAs) that could knock down SLY protein expression to undetectable levels and inserted these into mice. They found that the transgenic animals developed normally, but males were nearly sterile, with badly deformed sperm. Sperm are generated via meiosis, a special form of cell division that involves two consecutive rounds of cell division without an intervening bout of chromosome duplication. In this way, four sperm cells are created from one original cell; each new cell contains one copy of every autosome and either the X or the Y chromosome. As with regular cell division, during meiosis, all the chromosomes are packed up tightly for distribution among the new cells. Afterward, the new sperm's autosomes are unpacked for use. However, due to mechanisms that are poorly understood, transcription of most X- and Y-chromosome genes is maintained at a low level after meiosis. Multicopy genes, such as Sly, achieve a level of expression comparable to autosomal genes, perhaps because their extra copies make them more likely to be transcribed. Until now, the only thing known about this repression mechanism was that it might be regulated by genes residing on the Y chromosome. Mice that have a large deletion in the long (q) arm of the Y chromosome (known as MSYq-deficient animals) show less repression. Interestingly, the deletion in MSYq-deficient animals covers the region where the Sly gene copies are found, and results in morphological sperm defects that are similar to those observed in Sly-shRNA sperm. These observations led the authors to test whether Sly might be involved in the repression of sex chromosome genes after meiosis. Developing sperm cells are known to initially share their protein contents, and Cocquet et al. showed that SLY protein was indeed present in both X- and Y-chromosome–containing sperm in normal mice. Furthermore, consistent with the fact that SLY protein is predicted to have chromosome-binding activity, it was found near the sex chromosomes. This proximity led Cocquet et al. to examine whether loss of Sly might affect gene activity in sperm. In fact, they observed that many X- and Y-chromosome genes, and some autosomal genes, were more expressed (or de-repressed) in sperm from Sly-shRNA mice. Taken together, the authors' data indicate that Sly plays a central role in the post-meiotic repression of sex chromosome genes. Paradoxically, this means Sly represses its own expression (along with that of other genes). This represents an important insight both into the repression mechanisms and the function of this heretofore uncharacterized gene. Cocquet J, Ellis PJI, Yamauchi Y, Mahadevaiah SK, Affara NA, et al (2009) The Multi-Copy Gene Sly Represses the Sex Chromosomes in the Male Mouse Germline after Meiosis. doi: 10.1371/journal.pbio. 1000244 In male post-meiotic germ cells (that is, spermatids), SLY protein co-localizes with the X or the Y chromosome to repress transcription.

Cryptic improvement for fertility by continuous selfing of diploid potatoes using Sli gene

While a diploid potato was continuously selfed by a function of Sli gene, the level of heterozygosity reduced, and the fertility also reduced (inbreeding depression), which might be improved because continuous selfing could eliminate undesirable recessive alleles. To explore what was occurring as advancing self-generations in terms of fertility, 51 plants of an S3 diploid potato family were evaluated for fertility-related traits and analyzed by RFLP markers. The level of heterozygosity was positively correlated with any of fertility-related traits (r = 0.108−0.333). At least six loci on six chromosome sections were associated with fertility-related quantitative trait loci (QTLs), of which three performed better in the heterozygotes and three in the homozygotes. By further continued selfing, QTLs performing better in heterozygotes were likely fixed to homozygotes with secondly better performance, and those performing better in homozygotes were fixed to the best genotypes. Thus, the selfed progenies were cryptically being improved for fertility by genetic fixation to the best or better genotypes in fertility-related QTLs.

Expression of S-locus inhibitor gene (Sli) in various diploid potatoes

S-locus inhibitor gene (Sli), which can inhibit gametophytic self-incompatibility in diploid potatoes and alter self-incompatible to self-compatible plants, was introduced by crossing into 32 diploid genotypes as females and its expression in the F1 and S1 progenies was investigated. We found that the expression of self-compatibility in the F1 hybrid progeny depended largely upon the female genotypes and partly upon the male genotypes (=Sli gene donor clones). Successful females produced hybrid plants, in which 67.1% of self-pollinated plants set S1 seeds. By second selfing upon the S1 plants, an average of 44.2% of self-pollinated plants were self-compatible. Unsuccessful females produced hybrids, most of which were self-incompatible or male-sterile. Restriction fragment patterns of chloroplast DNA (ctDNA) were able to distinguish successful females (S- or A-type ctDNA) from unsuccessful females (W- or T-type ctDNA). A ctDNA high-resolution marker analysis using seven microsatellites and H3 marker supported a higher degree of differentiation between the two groups of ctDNA types and implied a possible interaction between the cytoplasm and Sli gene function. However, it has been known that the cytoplasm having T-type ctDNA and that derived from Solanum demissum (haplotype 26 of W-type ctDNA) cause male sterility, and the present case with unsuccessful females were likely caused by male sterility rather than the failure of Sli gene function.

The Effects of Deletions of the Mouse Y Chromosome Long Arm on Sperm Function—Intracytoplasmic Sperm Injection (ICSI)-Based Analysis1

In mouse and man, Y chromosome deletions are frequently associated with spermatogenic defects. XY(Tdy)(m1)qdelSry males have an extensive Yq deletion that almost completely abolishes the expression of two gene families, Ssty and Sly, located within the male-specific region of the mouse Y long arm. These males exhibit severe sperm defects and sterility. XY(RIII)qdel males have a smaller interstitial Yq deletion, removing approximately two thirds of Ssty/Sly gene copies, and display an increased incidence of mild sperm head anomalies with impairment of fertility and an intriguing distortion in the sex ratio of offspring in favor of females. Here we used intracytoplasmic sperm injection (ICSI) to investigate the functional capacity of sperm from these Yq deletion males. Any selection related to the ability of sperm to fertilize in vitro is removed by ICSI, and we obtained two generations of live offspring from the infertile males. Genotyping of ICSI-derived offspring revealed that the Y(Tdym1)qdel deletion does not interfere with production of Y chromosome-bearing gametes, as judged from the frequency of Y chromosome transmission to the offspring. ICSI results for XY(RIII)qdel males also indicate that there is no deficiency of Y sperm production in this genotype, although the data show an excess of females following in vitro fertilization and natural mating. Our findings suggest that 1) Yq deletions in mice do not bias the primary sex ratio and 2) Y(RIII)qdel spermatozoa have poorer fertilizing ability than their X-bearing counterparts. Thus, a normal complement of the Ssty and/or Sly gene families on mouse Yq appears necessary for normal sperm function. ICSI was successfully used to reproduce infertile mice with Yq deletions, and the analysis of sperm function in obtained offspring demonstrated that gene families located within the deletion interval are necessary for normal sperm function.

Toward the development of highly homozygous diploid potato lines using the self-compatibility controllingSligene

Cultivated diploid potatoes (2n = 2x = 24) are self-incompatible, but can be altered to become self-compatible using the Sli gene. Previously, a diploid clone 97H32-6 was selfed up to S3 using the Sli gene. To explore the usefulness of the Sli gene for the production of highly homozygous diploid potatoes, 2 S4 families from the above 97H32-6 derived S3 lines (inbred series A) and 3 S5 families by continuous selfings from a different F1 (= S0) plant (inbred series B) were developed. The level of heterozygosity and the location of heterozygous loci on the genetic map were investigated using RFLP and AFLP markers. The average heterozygosity levels of the originally heterozygous loci decreased from 100% in S0 to 10.7% in S4 and 8.6% in S5 (inbred series A and B, respectively). The average rate of reduction in heterozygosity per generation (38.4% and 38.5% for inbred series A and B, respectively) was lower than the theoretically expected rate (50%). However, none of the loci or chromosome sections was exclusively heterozygous in the advanced self-progeny. Thus, highly homozygous and seed-propagated diploid potatoes could be obtained by repeated selfing using the Sli gene.

Role of suilysin in pathogenesis of Streptococcus suis capsular serotype 2

Three suilysin (SLY) knockout mutant strains of Streptococcus suis serotype 2 were generated by allelic replacement from one North American and two European wild type strains. The mutants were characterized by Southern blot, Western blot and phenotyping. In vitro bactericidal testing showed that both wild type and SLY mutants were resistant to bactericidal factors in whole pig blood. To demonstrate the role of SLY during S. suis infection, four animal trials were carried out using young pigs. Either high dose (4×10 6 CFU/ml/pig) or low dose (0.5×10 6 CFU/ml/pig) live cell aerosol was applied to the pharynx. In one trial, a low challenge dose of North American strain SX332 and its isogenic sly − mutant strain (SX932) resulted in acute disease in 3/5 of pigs exposed to the wild type strain, while 5/5 of pigs exposed to the mutant strain survived the trial. In the repeat trial, 1/8 of pigs in wild type group and 6/8 of pigs in mutant group developed disease. The high dose trial with 332/932 pair showed that 4/8 pigs challenged with wild type and 5/8 of pigs challenged with mutant strain developed disease respectively. The third low dose trial, using European strain 31533 and its isogenic sly − mutant strain SX911, showed that 1/8 of pigs challenged with the wild type strain and 4/8 of pigs challenged with the corresponding mutant strain developed disease. All the diseased pigs showed fever, clinical signs and developed septicemia. S. suis was isolated from tissue samples such as brain, submandibular lymph node, lung, spleen, liver, heart or joint. Serum antibody titer against cell surface proteins changed little while the antibody titer against SLY increased only in the wild type group after challenge. sly gene was cloned and expressed in E. coli. The recombinant SLY (rSLY) protein showed 800 hemolysin units per μg protein. In vitro study showed that rSLY triggered TNFα production by human monocytes and IL-6 production by pig pulmonary alveolar macrophages and monocytes. Thus, the results of this study suggest that SLY does not seem to be a critical virulence factor for S. suis serotype 2 respiratory infection, but by stimulating cytokine release it may play a role in innate immunity.

Thermosensitive Suicide Vectors for Gene Replacement in Streptococcus suis

Three thermosensitive (Ts) suicide vectors, pSET4s, pSET5s, and pSET6s, have been constructed for gene replacement in Streptococcus suis. Each vector contains an antibiotic-resistance gene (spc or cat), a Ts replication origin of pWV01 lineage, multiple cloning sites, lacZ′, and the ColE1 replication origin of pUC19. These vectors could be propagated at 37°C in Escherichia coli, but their replication was blocked above 37°C in S. suis. Moreover, the thermosensitivity of the replication origin was confirmed in S. equi ssp. equi, S. equi ssp. zooepidemicus, and S. dysgalactiae by using pSET4s. For inactivation of the sly gene, which encodes a thiol-activated hemolysin of S. suis, pSLYK, in which the sly gene was interrupted by the cat gene, was constructed using pSET4s and introduced into S. suis DAT2. After growth at the nonpermissive temperature under the antibiotic pressure, the chromosomal sly gene was replaced with the sly::cat gene of pSLYK by a double-crossover event at a rate of 2.6% among chloramphenicol-resistant cells. Moreover, complementation of the sly gene by use of the previously reported S. suis-E. coli shuttle vector pSET2 was demonstrated. These results indicate that the Ts suicide vectors described here will facilitate the genetic analysis of S. suis and other streptococci of veterinary importance by means of allelic exchange of the genes of interest via homologous recombination.

Molecular cloning and characterization of a novel SH3 protein (SLY) preferentially expressed in lymphoid cells

A novel full-length cDNA was isolated from a murine T-cell lymphoma library that has an open reading frame encoding 381 amino acids. The predicted protein (termed SLY) contains a Src homology 3 domain and a sterile α motif, suggesting that it functions as a signaling adaptor protein in lymphocytes. Northern blot and in situ hybridization analysis showed a preferential expression in lymphoid tissues. The sly gene is located on the X-chromosome in close proximity to genes involved in various immune disorders. This is consistent with an additional role of SLY in immune pathology.

Production of inbred progenies of diploid potatoes using an S-locus inhibitor (Sli) gene, and their characterization.

To develop inbred lines from self-incompatible, cultivated diploid potatoes, an S-locus inhibitor (Sli) gene derived from a self-compatible variant of a wild potato species, Solanum chacoense, was incorporated into various cultivated diploid potatoes. The progeny was selfed twice by the action of the Sli gene to obtain 74 S2 inbred clones belonging to 8 families. More than 40% of them were either non-flowering or pollen sterile. Among the pollen fertile clones, self-compatible clones occurred with a much lower frequency (20.9%) than expected (83.3%). The result demonstrated that self-compatibility was introduced and expressed in the gene pool of cultivated diploid potatoes by an action of the Sli gene, although serious inbreeding depression associated with selfing occurred. The genotypes of S2 inbreds were surveyed using 46 S. chacoense-specific RFLP (restriction fragment length polymorphism) markers covering the whole potato genome. More than half of the markers (67.4%) showed distorted segregation. Particularly, all markers on chromosome 12 were overrepresented in the S2 inbreds. This confirms our earlier finding that the Sli gene locates on chromosome 12 and the alleles linked with this gene are preferentially transmitted because of its essential requirement for selfing.

Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates

Nineteen Streptococcus suis type 2 isolates that had been analyzed previously for hemolysin production, ribotype, and virulence in pigs were examined for presence of the gene coding for suilysin by PCR amplification, and southern blot and hybridization techniques. Based on southern blot and hybridization analysis, all isolates tested contained at least a portion of the suilysin gene. PCR amplification of the entire gene resulted in gene fragments from five of the seven highly virulent isolates and none of the moderately virulent or avirulent isolates. Additional PCR analysis showed that mutation or deletions at the 5′ end of the suilysin gene in the less virulent isolates prevented amplification of the sly gene fragment from those isolates. The MRP+ (muramidase-released protein) EF+ (extracellular protein) phenotype was also expressed by the same five highly virulent/ sly+ isolates.

Characterisation of the gene encoding suilysin from Streptococcus suis and expression in field strains

The gene encoding suilysin was cloned from Streptococcus suis serotype 2 strain P1/7. Analysis of the nucleotide and translated amino acid sequence confirmed suilysin to be a member of the thiol activated cytolysin group (TACY). The pneumolysin from Streptococcus pneumoniae is the most closely related orthologous gene known. Suilysin was overexpressed in E. coli in an active haemolytic form. A strong correlation between the presence of the sly gene and haemolytic activity in the supernatant of S. suis field strains was found. Of 158 strains tested, 63% contained the gene. Within the (most prevalent) serotype 2, the sly gene was demonstrated in 95% of the strains isolated in Eurasia, but only in 7% of the strains from North America.

Characterisation of the gene encoding suilysin from Streptococcus suis and expression in field strains.

The gene encoding suilysin was cloned from Streptococcus suis serotype 2 strain P1/7. Analysis of the nucleotide and translated amino acid sequence confirmed suilysin to be a member of the thiol activated cytolysin group (TACY). The pneumolysin from Streptococcus pneumoniae is the most closely related orthologous gene known. Suilysin was overexpressed in E. coli in an active haemolytic form. A strong correlation between the presence of the sly gene and haemolytic activity in the supernatant of S. suis field strains was found. Of 158 strains tested, 63% contained the gene. Within the (most prevalent) serotype 2, the sly gene was demonstrated in 95% of the strains isolated in Eurasia, but only in 7% of the strains from North America.

Genetics of self-compatibility in a self-incompatible wild diploid potato species Solanum chacoense. 2. Localization of an S locus inhibitor (Sli) gene on the potato genome using DNA markers

A self-compatible (SC) hybrid plant F1-1 was obtained from a cross between a SC variant of a wild diploid potato species, Solanum chacoense, and a self-incompatible (SI) cultivated diploid species, S. phureja. The clone F1-1 has previously been proposed to have a dominant S locus inhibitor gene (Sli) in a heterozygous condition. It was crossed as a male parent with a selected clone from a S. stenotomum-S. phureja population, resulting in a segregating population consisting of 116 hybrid plants. Self-compatibility was assessed by selfing each of the hybrids. Sixty-six of them were SC, while 35 were SI, showing a significant distortion from an expected Mendelian ratio of 1:1. A genetic linkage map was constructed using DNA markers to localize the Sli gene. A total of 28 RAPD and 127 RFLP markers identified 109 mapping positions on 12 linkage groups. The Sli gene was mapped at a distal end of chromosome 12. Since the S locus has been localized on chromosome 1 on the potato RFLP map, it is confirmed that the Sli gene is independent of the S locus.

Genetics of self-compatibility in a self-incompatible wild diploid potato species Solanum chacoense. 1. Detection of an S locus inhibitor (Sli) gene

A self-compatible (SC) variant of a wild diploid potato species, Solanum chacoense, which is normally self-incompatible (SI), was investigated for the nature and genetics of self-compatibility. It was crossed with a SI cultivated diploid potato species, S. phureja. The F1 progeny segregated SC vs. SI. Diallel crosses were made among 15 F1's. Self-compatibility was tested in a selfed family of a parental SC variant and in sib-mated and selfed families of F1 progeny. All the data suggest that there is a single dominant gene (Sli) with sporophytic action inhibiting S gene expression in the pollen. Plants having a ‘Sli’ gene, produce pollen which is compatible to its own parent and plants with similar S genes. The ‘Sli’ gene has been maintained in a heterozygous condition through eight selfing generations (S8) implying that dominant homozygotes might be associated with lethality.

The Sip(Sli) gene of Streptomyces lividans TK24 specifies an unusual signal peptidase with a putative C-terminal transmembrane anchor.

Type I signal peptidases (SPases) are a widespread family of enzymes which remove signal peptides from proteins translocated across cellular membranes. Here, we report the first isolation of a gene coding for type I signal peptidase of Streptomyces, denoted Sip(Sli). The sip(sli) gene specifies a protein of 291 amino acids. Thus Sip(Sli) is much larger (approximately 100 amino acids) than other known SPases of Gram-positive bacteria and resembles SPases of Gram-negative bacteria, showing the highest degree of similarity to an SPase of the cyanobacterium Phormidium laminosum. Sip(Sli) contains conserved serine and lysine residues, which are believed to be required for the catalytic activity. Similar to other known SPases from Gram-positive bacteria, Sip(Sli) seems to have only one N-terminal transmembrane anchor. In addition, Sip(Sli) seems to contain a second transmembrane anchor at the C-terminus, which is an unusual feature for type I signal peptidases.

Calcium and SLY Genes Suppress the Temperature-Sensitive Secretion Defect of Saccharomyces cerevisiae uso1 Mutant

Saccharomyces cerevisiae uso1-1 mutant stops the transport of secretory proteins from the endoplasmic reticulum to the Golgi apparatus at 37°C. We found that this temperature-sensitive defect was suppressed either by increasing the concentration of calcium ion in the medium or by introducing in the cell the SLY genes which suppress the defect of Ypt1 protein, a small GTP-binding protein. The common phenotype and suppression of the mutants suggest that Uso1 and Ypt1 proteins function in the same process of protein transport, i.e., targeting or fusion of the transport vesicles to the Golgi membrane.

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

The Yeast SLY Gene Products, Suppressors of Defects in the Essential GTP-Binding Ypt1 Protein, May Act in Endoplasmic Reticulum-to-Golgi Transport

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily.

In Saccharomyces cerevisiae, the GTP-binding Ypt1 protein (Ypt1p) is essential for endoplasmic reticulum-to-Golgi protein transport. By exploiting a GAL10-YPT1 fusion to regulate YPT1 expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLY1-20, that allowed YPT1-independent growth were isolated. Wild-type Sly1p is hydrophilic, is essential for cell viability, and differs from Sly1-20p by a single amino acid. SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes. Sly41p is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator. SLY12 but not SLY41 is an essential gene. The SLY2 null mutant is cold and heat sensitive. The SLY gene products may comprise elements of the protein transport machinery.