Toshiko Ohta

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.

Primary structure of the β‐subunit of Torpedo californica (Na+ + K+)‐ATPase deduced from the cDNA sequence

DNA complementary to the Torpedo californica electroplax mRNA coding for the β‐subunit of (Na+ + K+)‐ATPase has been cloned by screening a cDNA library with an oligodeoxyribonucleotide probe. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 305 amino acid residues (including the initiating methionine). The transmembrane topology and the potential N‐glycosylation sites of this polypeptide are discussed.

Immunohistochemical localization of Na+, K+-ATPase in the choroid plexus

To determine if canine and rat choroid plexus Na+,K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified canine kidney medulla Na+,K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified canine kidney Na+,K+-ATPase and canine kidney microsomes were treated with antiserum followed by [125I]protein A and autoradiography, the canine microsomes and purified Na+,K+-ATPase showed a prominent radioactive band coincident with the alpha-, beta- and gamma-subunits of the purified canine kidney enzyme. When the rabbit immunoglobulin that was purified from the Na+,K+-ATPase antiserum through DEAE-cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus fixed with Bouins fixative, intense immunoreactive staining was present on the epithelial cells of both choroid plexuses but was not found in the tissue around the vessel. The staining was especially confined to apical surfaces of the epithelial cells. The same procedure was performed in the canine kidney, and immunostaining was obtained in the tubules where Baskin and Stahl described the enzyme localization. No staining was seen with pre-immune serum of the normal rabbit. We concluded that both the canine and rat choroid plexus are rich in Na+,K+-ATPase, which plays an important role in cerebrospinal fluid (CSF) secretion.

A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes

Background:  Staphylococcus aureus is a major human pathogen and causes a serious hospital infection due to the acquired multidrug resistance. Unlike the well‐studied bacteria such as Escherichia coli and Bacillus subtilis, which have seven and 18 sigma factors, respectively, only two sigma factors have been known for S. aureus. We searched for possible sigma factor genes by examining the S. aureus genome with a special attention to the gene arrangement around the sigma factor genes of a close relative, B. subtilis.

Structural basis for multimeric heme complexation through a specific protein-heme interaction: the case of the third neat domain of IsdH from Staphylococcus aureus.

To elucidate the heme acquisition system in pathogenic bacteria, we investigated the heme-binding properties of the third NEAT domain of IsdH (IsdH-NEAT3), a receptor for heme located on the surfaces of pathogenic bacterial cells, by using x-ray crystallography, isothermal titration calorimetry, examination of absorbance spectra, mutation analysis, size-exclusion chromatography, and analytical ultracentrifugation. We found the following: 1) IsdH-NEAT3 can bind with multiple heme molecules by two modes; 2) heme was bound at the surface of IsdH-NEAT3; 3) candidate residues proposed from the crystal structure were not essential for binding with heme; and 4) IsdH-NEAT3 was associated into a multimeric heme complex by the addition of excess heme. From these observations, we propose a heme-binding mechanism for IsdH-NEAT3 that involves multimerization and discuss the biological importance of this mechanism.

Staphylococcus aureus requires cardiolipin for survival under conditions of high salinity

BackgroundThe ability of staphylococci to grow in a wide range of salt concentrations is well documented. In this study, we aimed to clarify the role of cardiolipin (CL) in the adaptation of Staphylococcus aureus to high salinity.ResultsUsing an improved extraction method, the analysis of phospholipid composition suggested that CL levels increased slightly toward stationary phase, but that this was not induced by high salinity. Deletion of the two CL synthase genes, SA1155 (cls1) and SA1891 (cls2), abolished CL synthesis. The cls2 gene encoded the dominant CL synthase. In a cls2 deletion mutant, Cls1 functioned under stress conditions, including high salinity. Using these mutants, CL was shown to be unnecessary for growth in either basal or high-salt conditions, but it was critical for prolonged survival in high-salt conditions and for generation of the L-form.ConclusionsCL is not essential for S. aureus growth under conditions of high salinity, but is necessary for survival under prolonged high-salt stress and for the generation of L-form variants.

Expression of a Cryptic Secondary Sigma Factor Gene Unveils Natural Competence for DNA Transformation in Staphylococcus aureus

It has long been a question whether Staphylococcus aureus, a major human pathogen, is able to develop natural competence for transformation by DNA. We previously showed that a novel staphylococcal secondary sigma factor, SigH, was a likely key component for competence development, but the corresponding gene appeared to be cryptic as its expression could not be detected during growth under standard laboratory conditions. Here, we have uncovered two distinct mechanisms allowing activation of SigH production in a minor fraction of the bacterial cell population. The first is a chromosomal gene duplication rearrangement occurring spontaneously at a low frequency [≤10−5], generating expression of a new chimeric sigH gene. The second involves post-transcriptional regulation through an upstream inverted repeat sequence, effectively suppressing expression of the sigH gene. Importantly, we have demonstrated for the first time that S. aureus cells producing active SigH become competent for transformation by plasmid or chromosomal DNA, which requires the expression of SigH-controlled competence genes. Additionally, using DNA from the N315 MRSA strain, we successfully transferred the full length SCCmecII element through natural transformation to a methicillin-sensitive strain, conferring methicillin resistance to the resulting S. aureus transformants. Taken together, we propose a unique model for staphylococcal competence regulation by SigH that could help explain the acquisition of antibiotic resistance genes through horizontal gene transfer in this important pathogen.

Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the α-subunit

Currents generated by the Na+/K+ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na+] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had α-subunits truncated before position Lys28 (αΔK28) or Thr29 (αΔT29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K+ show voltage-dependent inhibition by external [Na+]. The apparent K1 values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na+-K+ competition for external binding to the E2P form, apparent K1 values and effective charges for the interaction of the Na+ ions with the E2P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K1 value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K1 value. For αΔK28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS28 is included in the truncation (α·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K1 value is reduced to 107 mM. The K, values for pump inhibition by external Na+, calculated by taking into account Na+-K+ competition, are smaller than the K/12 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km, values. The variations of the parameters describing inhibition by external [Na+] are qualitatively similar to those described for the stimulation of the pumps by external [K+] in the absence of extracellular [Na+]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na+ and K+ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.

Methicillin-Resistant Staphylococcus saprophyticus Isolates Carrying Staphylococcal Cassette Chromosome mec Have Emerged in Urogenital Tract Infections

ABSTRACT Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed β-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.