K2 Strains Research Articles

L-A-lus, a New Variant of the L-A Totivirus Found in Wine Yeasts with Klus Killer Toxin-Encoding Mlus Double-Stranded RNA: Possible Role of Killer Toxin-Encoding Satellite RNAs in the Evolution of Their Helper Viruses

Yeast killer viruses are widely distributed in nature. Several toxins encoded in double-stranded RNA (dsRNA) satellites of the L-A totivirus have been described, including K1, K2, K28, and Klus. The 4.6-kb L-A genome encodes the Gag major structural protein that forms a 39-nm icosahedral virion and Gag-Pol, a minor fusion protein. Gag-Pol has transcriptase and replicase activities responsible for maintenance of L-A (or its satellite RNAs). Recently we reported a new killer toxin, Klus. The L-A virus in Klus strains showed poor hybridization to known L-A probes, suggesting substantial differences in their sequences. Here we report the characterization of this new L-A variant named L-A-lus. At the nucleotide level, L-A and L-A-lus showed only 73% identity, a value that increases to 86% in the amino acid composition of Gag or Gag-Pol. Two regions in their genomes, however, the frameshifting region between Gag and Pol and the encapsidation signal, are 100% identical, implying the importance of these two cis signals in the virus life cycle. L-A-lus shows higher resistance than L-A to growth at high temperature or to in vivo expression of endo- or exonucleases. L-A-lus also has wider helper activity, being able to maintain not only Mlus but also M1 or a satellite RNA of L-A called X. In a screening of 31 wine strains, we found that none of them had L-A; they carried either L-A-lus or a different L-A variant in K2 strains. Our data show that distinct M killer viruses are specifically associated with L-As with different nucleotide compositions, suggesting coevolution.

Capsular Polysaccharide Synthesis Regions in Klebsiella pneumoniae Serotype K57 and a New Capsular Serotype

Community-acquired pyogenic liver abscess caused by Klebsiella pneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype.

Strains of Lentinula edodes suppress growth of phytopathogenic fungi and inhibit Alagoas serotype of vesicular stomatitis virus

Four Lentinula edodes strains (Le10, 46, K2, Assai) were assessed for their antagonistic effect on four filamentous fungus species of agricultural importance (Helminthosporium euphorbiae, Helminthosporium sp, Fusarium solani and Phomopsis sojae) and on Alagoas serotype of Vesicular Stomatitis Virus (VSA). The L. edodes strains studied had variable effects on the filamentous fungi and on VSA. The K2 and Le10 strains were antagonistic on the fungi assessed and the 46 and K2 strains were efficient on the Vesicular Stomatitis Virus. The results widened the list of beneficial effects of L. edodes on the control and prevention of animal pathogenic virus and filamentous fungi.

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations.

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.

Presence of non-supressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.

Relationships among capsular structure, phagocytosis, and mouse virulence in Klebsiella pneumoniae

Klebsiella pneumoniae strains of the K2 capsular serotype are usually highly virulent in mice, which is in contrast to the low virulence of most other serotypes. Here we used a genetic approach to examine the relative contribution of capsule type to the virulence of K. pneumoniae in mice. We used wild-type strains expressing capsular polysaccharide (CPS) serotypes K2 (strain KPA1) and K21a (strains KPB1 and KPC1), which were then used to construct capsule-switched derivatives. The close proximity of the cps gene cluster to selectable his markers made it possible to mobilize the cps genes by conjugation from one serotype (donor) to another (recipient) and to obtain recombinants in which interserotype switching had occurred by reciprocal recombination. Each capsule-switched derivative examined of the KPA and KPC strain backgrounds produced a CPS that was immunologically and structurally identical to that of the donor. Strain background was confirmed by demonstrating restriction fragment length polymorphism patterns identical to those of the respective recipients. The parent strains were then compared with capsule-switched recombinants for phenotypic properties associated with virulence. Clearance from the bloodstreams of mice was rapid in serotype K21a strains of either wild-type or recombinant origin, whereas K2 strains remained viable in the blood during the period examined. These differences appeared to be dependent upon the CPS type but independent of strain background. Binding to macrophages was higher in K21a strains than in those with the K2 capsule and was also independent of the strain background. Both blood clearance and macrophage-binding activities were completely inhibited by yeast mannan, suggesting that they were mediated via the macrophage mannose receptor. The K2 parent strain was highly virulent to mice (50% lethal dose [LD50], 3 x 10(3)), while the K21a parent strains demonstrated low virulence (LD50, > 2 x 10(8)). Interestingly, the virulence of recombinant KPC10(cpsK2), originally of the KPC1(cpsK21a) background, was intermediate (LD50, 4 x 10(5)). In contrast, both cpsK21a recombinants of the originally virulent KPA1 (cpsK2) background became nearly avirulent (LD50, > 2 x 10(8)). Six additional serotypes (K12, K24, K32, K55, K62, and K67) were examined, and all showed a positive correlation between the ability of the Klebsiella serotype to interact with a human mannose receptor, as expressed by Cos I cell recombinants, and the LD50 of the serotype. These results suggest that expression of a capsule which is recognized by the mannose receptor markedly affects the interaction with macrophages and blood clearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of extracapsular polysaccharide synthesis in Klebsiella pneumoniae by RmpA2, which shows homology to NtrC and FixJ

We determined the complete nucleotide sequence of a 2.1-kb HindIII-EcoRI fragment that was cloned from a resident large plasmid of Klebsiella pneumoniae Chedid, a highly virulent and mucoviscous strain of the O1:K2 serotype. This fragment encoded an ability to enhance K2 capsular polysaccharide synthesis in K. pneumoniae, and a 636-bp open reading frame (rmpA2) was found. The 411-bp rmpA reported to be involved in the virulence and mucoid phenotypes of K. pneumoniae by Nassif et al. (Mol. Microbiol. 3:1349-1359, 1989) was a part of rmpA2. Eighty percent homology in nucleotide sequence was found between rmpA2 and rmpA in the corresponding regions. The central domain of the deduced amino acid sequence of RmpA2 showed considerable homology to the central domains of NtrC of K. pneumoniae and Escherichia coli, to which the sigma factor of RNA polymerase binds. The C-terminal domain of RmpA2 also demonstrated considerable homology with the putative helix-turn-helix motifs of LuxR of Vibrio fischeri and FixJ of Rhizobium meliloti. Moreover, RmpA2 also showed some homology in its N- and C-terminal regions to those of RcsA, a transcriptional activator for colanic acid synthesis in E. coli. On the other hand, a sequence upstream of rmpA2 was found to be highly homologous to insertion sequence 3 of members of the family Enterobacteriaceae. Southern hybridization analysis suggested that rmpA2 exists on the large plasmids of all mucoviscous virulent K2 strains but not on those of the slightly mucoviscous avirulent strains. Freeze substitution electron microscopy and fluorescent-antibody staining with anti-K2 serum revealed that K. pneumoniae Chedid has a dense and thick capsule (180 nm) with dense extracapsular substance, whereas K. pneumoniae K2-215, one of the slightly mucoviscous and avirulent strains, has a capsule which is looser and thinner (120 nm) than that of strain Chedid and no extracapsular substance. Introduction of rmpA2 into K2-215 as well as reference strains K. pneumoniae K9 and K72 resulted in a change of the colony phenotype to highly mucoviscous through abundant production of extracapsular substance which reacted with anti-K2, -K9, or -K72, respectively, as did their parental strains. From these results, it is suggested that RmpA2 belongs to the family of transcriptional regulators and confers a highly mucoviscous phenotype on cells of various serotypes of K. pneumoniae by enhancing extracapsular polysaccharide synthesis.

Interaction of Klebsiella capsule type 7 with human polymorphonuclear leucocytes

Klebsiella serotype K7 is found among the capsule types that are most prevalent in respiratory tract isolates. To evaluate the significance of the K7 antigen in bacteria-leucocyte interactions, K7-encapsulated Klebsiella pneumoniae strains and their non-capsulate mutants were investigated. The K7 isolates were compared to K2-capsulate strains and their respective K derivatives. K7-capsulate bacteria were less hydrophilic, and more readily phagocytosed and killed by human polymorphonuclear leucocytes (PMNL) than K2 strains. Loss of the K7 antigen resulted in increased surface hydrophobicity but did not affect phagocytosis and killing, whereas loss of the K2 capsule caused greater susceptibility to the phagocytic and killing action of PMNL. Both the K7 and K2 antigen stimulated the extracellular release of lysozyme from neutrophils but not of myeloperoxidase, indicating degranulation of only secondary granules. All K − mutants induced the release of both lysozyme and myeloperoxidase. Our results suggest that, in contrast to the K2 antigen, the K7 capsular polysaccharide does not confer antiphagocytic properties on bacteria. However, the K7 antigen is able to impede the extracellular release of primary granule enzymes.

Klebsiella capsular type K7 in relation to toxicity, susceptibility to phagocytosis and resistance to serum.

Klebsiella strains possessing capsule type K7 are found predominantly in respiratory secretions. To investigate the importance of this K antigen in virulence, 13 K7 strains were compared with K2 capsulate isolates which are generally regarded as highly virulent. The toxicity of the strains was determined in a mouse peritonitis model. Generally, K7 isolates were significantly less toxic for mice than K2 strains. In the absence of serum, neither capsule type showed much stimulation of leucocytes, measured as the chemiluminescence (CL) response of human polymorphonuclear leucocytes (PMNL). However, in the presence of normal human serum, CL values with K7 strains increased considerably, whereas the CL response to K2 isolates was unaffected. Correspondingly, intracellular killing by PMNL was observed with K7 strains only, whereas K2 isolates proved to be relatively resistant to phagocytic destruction. No correlation was found between capsule type K7 and serum resistance. These data suggest that, in contrast to K2, capsule type K7 may not be a critical factor in the virulence of K7-capsulate Klebsiella strains nor does it seem to act as an antiphagocytic barrier.

Size differentiation of M 2 genomes among K 2 killer yeasts

Viral double-stranded ribonucleic acid (dsRNA) genomes from killer yeasts isolated from wineries were extracted and compared with that of reference K2 and K3 killer strains. The sizes of the L genomes of all the strains were similar but the M genomes varied from 1·3 to 1·5 kilobase pairs (kb). This incorporates the sizes attributed to the M genomes of K2 and K3 killer yeasts.

A murine monoclonal antibody against Klebsiella capsular polysaccharide is opsonic in vitro and protects against experimental Klebsiella pneumoniae infection

Murine monoclonal antibodies (mAb) of the immunoglobulin M class specific for the K2 capsular polysaccharide (CPS) of Klebsiella were isolated. One such mAb, termed III/5-1, was selected for further study. This mAb promoted the uptake and killing of Klebsiella pneumoniae K2 strains by human granulocytes and activated complement after contact with the bacteria. The efficiency of mAb-mediated phagocytosis and complement activation was inversely related to the amount of capsular material produced by the test strain. MAb III/5-1 was found to be effective at preventing fatal experimental K. pneumoniae K2 sepsis when administered prophylactically, the degree of protection being dependent upon the amount of CPS produced by the challenge strain.

Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae.

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.

New bacteriophage typing scheme for subdivision of the frequent capsular serotypes of Klebsiella spp.

A bacteriophage typing scheme for hospital isolates of Klebsiella spp. was developed. The scheme was designed specifically as a secondary typing method to discriminate between strains of serotypes K2, K3, and K21 but proved to be an efficient general typing method for strains of most serotypes. The set of 15 phages gave 87.3% typeability on 236 strains of more than 70 different serotypes. Typeability within the K2, K3, and K21 strains was 93, 89, and 91%, respectively. There was a mean of 3.2 reactions strain-1 for all phage-typeable strains. Of the serologically nontypeable strains, 76.7% were susceptible to one or more phages. The most common pattern accounted for only 7% of the strains. The lytic patterns were reproducible if strains were typed on the same day, but differences were observed if strains were stored for 1 week or more before retyping. A total of 96.5% of the strains were typeable by a combination of capsular serology and phage typing.

On the mechanism of exclusion of M2 double-stranded RNA by L-A-E double-stranded RNA in Saccharomyces cerevisiae.

L-A-E double-stranded RNA (dsRNA), when introduced into cells carrying L-A-H and M2 dsRNAs, does not eliminate the L-A-H dsRNA, but (i) L-A-E does lower the copy number of L-A-H dramatically and (ii) L-A-E eliminates M2 dsRNA from the cell. That these two effects of L-A-E are related is shown by the fact that mutants of a strain carrying L-A-H and M2 selected for their resistance to exclusion of M2 by L-A-E [effect (ii)] have an altered L-A-H whose copy number is not lowered by L-A-E [effect (i)]. Although the L-A in K1 strains (L-A-HN in all cases examined) differs significantly both genetically and physically from the L-A in the K2 strain studied (L-A-H), the L-A-HN from the K1 strains can maintain M2 dsRNA, and the L-A-H from the K2 strains can maintain M1 dsRNA.

Chemiluminescence of human leukocytes stimulated by clinical isolates of Klebsiella.

Chemiluminescence (CL) of human polymorphonuclear leukocytes was determined after stimulation with 89 clinical isolates of Klebsiella which differed in serotype and in their virulence for mice. With K1, K2, K4 and K5 strains, a significantly lower CL response was observed than with K3, K6 and K greater than 6 strains. These results correlated well with virulence: greater virulence could be explained by greater resistance to phagocytosis.

Virulence of Klebsiella strains in experimentally induced skin lesions in the mouse.

The virulence of 93 clinical isolates of Klebsiella was compared in a mouse model by subcutaneous injection. Skin pathogenicity was measured by estimating the number of viable bacteria in the lesions 24 h after infection with a dose of 10(7) bacteria. Strains of serotypes K1-6 were compared with strains of serotypes higher than K6. All K1 and K5, and some K2 and K4 strains were more virulent for mice than strains with a serotype higher than K6. The K3 strains were significantly less virulent than the strains with a serotype higher than K6. The bacteriological findings were confirmed by histological examination with some strains. No differences in virulence were observed between strains of the same serotype isolated from patients with cystitis or from those with pyelonephritis, nor between strains of the same serotype isolated from the blood of patients with septicaemia or from other sites. The mouse model has been found satisfactory for observing differences in virulence between Klebsiella isolates.

Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types.

Eighty-two Klebsiella O1 strains belonging to various K types were tested for virulence for mice by intraperitoneal inoculation. They comprised 49 strains newly isolated from various clinical specimens, 31 reference strains, and 2 laboratory strains. Of 9 Klebsiella O1:K2 strains, 7 were highly virulent inasmuch as their 50% lethal doses per mouse were less than 10 CFU, whereas the other 2 strains were avirulent even when encapsulated just like the virulent strains as revealed by the quellung test. Klebsiella K2 reference strain B5055 and strain Chedid, which were maintained in vitro for a long time, were highly virulent. Of 8 Klebsiella O1:K1 strains, 2 showed moderate virulence, and the other 6 strains had relatively low or no virulence. We found no definite correlation between the virulence of Klebsiella K1 and K2 strains for mice and the kind of clinical specimens in which they originated. All of the Klebsiella O1 strains tested belonging to K types other than K1 and K2 showed very low or no virulence for mice.

Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN.

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.