Tsutomu Sekizaki

Significant contribution of the pgdA gene to the virulence of Streptococcus suis

Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S.u2003suis peptidoglycan (PG) and found, among other modifications, N‐deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N‐deacetylation correlated with absence of significant lysozyme resistance when wild‐type S.u2003suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S.u2003suis may enhance PG N‐deacetylation under these conditions. Evaluation of the ΔpgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S.u2003suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the ΔpgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N‐deacetylation is an important factor in S.u2003suis virulence.

CROSS-TALK TO THE GENES FOR BACILLUS ANTHRACIS CAPSULE SYNTHESIS BY ATXA, THE GENE ENCODING THE TRANS-ACTIVATOR OF ANTHRAX TOXIN SYNTHESIS

The two major virulence factors of Bacillus anthracis are the tripartite toxin and the polyglutamate capsule, which are encoded by genes on the large plasmids, pX01 and pX02, respectively. The genes atxA, located on pX01, and acpA, located on pX02, encode positive frans‐acting proteins that are involved in bicarbonate‐mediated regulation of toxin and capsule production, respectively. A derivative strain cured of pX01 produced less capsular substance than the parent strain harbouring both pX01 and pX02, and electroporation of the strain cured of pX01 with a plasmid containing the cloned atxA gene resulted in an increased level of capsule production. An acpA‐null mutant was complemented by not only acpA but also the atxA gene. The cap region, which is essential for encapsulation, contains three genes capB, capC, and cap A, arranged in that order. The atxA gene stimulated capsule synthesis from the cloned cap region. Transcriptional analysis of cap by RNA slot‐blot hybridization and primer‐extension analysis revealed that atxA activated expression of cap in trans at the transcriptional level. These results indicate that cross‐talk occurs, in which the pX01‐located gene, atxA, activates transcription of the cap region genes located on pX02. We identified two major apparent transcriptional start sites, designated P1 and P2, located at positions 731 bp and 625 bp, respectively, upstream of the translation‐initiation codon of capB. Transcription initiated from P1 and P2 was activated by both atxA and acpA, and activation appeared to be stimulated by bicarbonate. Deletion analysis of the upstream region of the cap

Genetic organization and preferential distribution of putative pilus gene clusters in Streptococcus suis.

Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A-L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.

Effect of growth temperature on maintenance of virulent Rhodococcus equi

Repeated passage of virulent Rhodococcus equi ATCC 33701 and L1 at 38 degrees C resulted in attenuation of the strains as a result of curing the virulence plasmid; at 30 degrees C, repeated passage had no such effect. At a temperature of 38 degrees C the plasmid-bearing cells replicated more slowly than their plasmid-cured derivatives and so were gradually replaced by cells lacking plasmids. In contrast, at a temperature of 30 degrees C the growth rate of either strain was not affected by the presence or absence of the plasmid. No plasmid-cured derivative was recovered from mouse organs at 48 h after inoculation of a mixture of equal numbers of bacteria with and without plasmids. It is concluded that under nonselective conditions growth temperature is an important factor in maintaining the virulence of R. equi.

Allelic variation and prevalence of serum opacity factor among the Streptococcus suis population

Serum opacity factor of Streptococcus suis (OFS) has recently been identified as a virulence determinant of an S. suis strain. In this study, we investigated the prevalence and variations of the ofs gene among 108 S. suis isolates from diseased and healthy pigs, and human patients. PCR screening and sequencing analysis showed that besides the ofs gene reported already (designated type 1), there were three allelic variants of ofs (designated types 2 to 4). Type-1 and type-2 ofs genes were expected to encode functional OFS, and SDS extracts of the isolates with type-1 ofs and type-2 ofs opacified horse serum. Culture supernatants of the isolates with type-2 ofs also showed strong serum opacification activity. In contrast, type-3 ofs was interrupted by a point mutation and type-4 ofs was disrupted by either insertion of an IS element or genetic rearrangement, and therefore the SDS extracts and culture supernatants of the isolates with type-3 ofs and type-4 ofs did not show serum opacification activity. Regardless of their origins, approximately 30 % of the isolates possessed functional OFSs, although type-2 ofs was found only in three isolates from healthy pigs. Multilocus sequence typing analysis showed that most of the isolates with type-1 ofs belonged to the sequence type (ST)1 complex, and most of the isolates with type-3 ofs and type-4 ofs belonged to the ST27 complex. The isolates with type-2 ofs were not assigned to a major ST complex. These results suggest that type-1 OFS contributes to the virulence of a limited number of S. suis isolates, i.e. those of the ST1 complex type, whereas other S. suis may not possess this category of virulence factor; the importance of type-2 OFS is obscure.