Chikara Kaito

Whole genome sequencing of meticillin-resistant Staphylococcus aureus

BACKGROUND Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Quantitative Evaluation of the Therapeutic Effects of Antibiotics Using Silkworms Infected with Human Pathogenic Microorganisms

ABSTRACT The injection of bacteria (Staphylococcus aureus, Stenotrophomonas maltophilia) or true fungi (Candida albicans, Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.

Silkworm pathogenic bacteria infection model for identification of novel virulence genes.

Silkworms are killed by injection of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, into the haemolymph. Gene disruption mutants of S. aureus whose open reading frames were previously uncharacterized and that are conserved among bacteria were examined for their virulence in silkworms. Of these 100 genes, three genes named cvfA, cvfB, and cvfC were required for full virulence of S. aureus in silkworms. Haemolysin production was decreased in these mutants. The cvfA and cvfC mutants also had attenuated virulence in mice. S. pyogenes cvfA‐disrupted mutants produced less exotoxin and had attenuated virulence in both silkworms and mice. These results indicate that the silkworm‐infection model is useful for identifying bacterial virulence genes.

Colony Spreading in Staphylococcus aureus

Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.

Transcription and Translation Products of the Cytolysin Gene psm-mec on the Mobile Genetic Element SCCmec Regulate Staphylococcus aureus Virulence

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus.

Abstract. We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the α-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) is a socially problematic pathogen that infects healthy individuals, causing severe disease. CA-MRSA is more virulent than hospital associated-MRSA (HA-MRSA). The underlying mechanism for the high virulence of CA-MRSA is not known. The transcription product of the psm-mec gene, located in the mobile genetic element SCCmec of HA-MRSA, but not CA-MRSA, suppresses the expression of phenol-soluble modulin α (PSMα), a cytolytic toxin of S. aureus. Here we report that psm-mec RNA inhibits translation of the agrA gene encoding a positive transcription factor for the PSMα gene via specific binding to agrA mRNA. Furthermore, 25% of 325 clinical MRSA isolates had a mutation in the psm-mec promoter that attenuated transcription, and 9% of the strains had no psm-mec. In most of these psm-mec-mutated or psm-mec-deleted HA-MRSAs, PSMα expression was increased compared with strains carrying intact psm-mec, and some mutated strains produced high amounts of PSMα comparable with that of CA-MRSA. Deletion of psm-mec from HA-MRSA strains carrying intact psm-mec increased the expression of AgrA protein and PSMα, and virulence in mice. Thus, psm-mec RNA suppresses MRSA virulence via inhibition of agrA translation and the absence of psm-mec function in CA-MRSA causes its high virulence property.

Novel DNA binding protein SarZ contributes to virulence in Staphylococcus aureus

We previously reported that the cvfA gene is a virulence regulatory gene in Staphylococcus aureus. Here, we identified a novel gene named sarZ that acts as a multicopy suppressor of decreased haemolysin production in the cvfA deletion mutant. The amount of sarZ transcripts was decreased in the cvfA mutant. The sarZ‐deletion mutant produced less haemolysin and attenuated virulence in a silkworm‐infection model and a mouse‐infection model. The amino acid sequence of the sarZ gene product had 19% identity with the transcription factor MarR in Escherichia coli, and the internal region contained a winged helix–turn–helix motif (wHTH), a known DNA binding domain. Purified recombinant SarZ protein had binding affinity for the promoter region of the hla gene that encodes α‐haemolysin. SarZ mutant proteins with an amino acid substitution in the N‐terminal region or in the wHTH motif had significantly decreased DNA binding. The mutated sarZ genes encoding SarZ mutant proteins with a low affinity for DNA did not complement the decreased haemolysin production or the attenuated killing ability against silkworms in the sarZ mutant. These results suggest that the DNA binding activity of the SarZ protein is required for virulence in S. aureus.

A Novel Gene, fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.

Silkworm apolipophorin protein inhibits Staphylococcus aureus virulence.

Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding α-hemolysin, hlb encoding β-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin.