Miki Matsuo

Mutated Response Regulator graR Is Responsible for Phenotypic Conversion of Staphylococcus aureus from Heterogeneous Vancomycin-Intermediate Resistance to Vancomycin-Intermediate Resistance

ABSTRACT Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

Pleiotropic Roles of Polyglycerolphosphate Synthase of Lipoteichoic Acid in Growth of Staphylococcus aureus Cells

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus Delta ltaS mutant could grow at 30 degrees C but not at 37 degrees C. Even at the permissive temperature, Delta ltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of Delta ltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the Delta ltaS mutant at 37 degrees C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the Delta ltaS mutation was found to be synthetically lethal with the Delta tagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30 degrees C, suggesting that LTA and WTA compensate for one another in an essential function.

Ordered Patterns of Cell Shape and Orientational Correlation during Spontaneous Cell Migration

Background In the absence of stimuli, most motile eukaryotic cells move by spontaneously coordinating cell deformation with cell movement in the absence of stimuli. Yet little is known about how cells change their own shape and how cells coordinate the deformation and movement. Here, we investigated the mechanism of spontaneous cell migration by using computational analyses. Methodology We observed spontaneously migrating Dictyostelium cells in both a vegetative state (round cell shape and slow motion) and starved one (elongated cell shape and fast motion). We then extracted regular patterns of morphological dynamics and the pattern-dependent systematic coordination with filamentous actin (F-actin) and cell movement by statistical dynamic analyses. Conclusions/Significance We found that Dictyostelium cells in both vegetative and starved states commonly organize their own shape into three ordered patterns, elongation, rotation, and oscillation, in the absence of external stimuli. Further, cells inactivated for PI3-kinase (PI3K) and/or PTEN did not show ordered patterns due to the lack of spatial control in pseudopodial formation in both the vegetative and starved states. We also found that spontaneous polarization was achieved in starved cells by asymmetric localization of PTEN and F-actin. This breaking of the symmetry of protein localization maintained the leading edge and considerably enhanced the persistence of directed migration, and overall random exploration was ensured by switching among the different ordered patterns. Our findings suggest that Dictyostelium cells spontaneously create the ordered patterns of cell shape mediated by PI3K/PTEN/F-actin and control the direction of cell movement by coordination with these patterns even in the absence of external stimuli.

Mutation of RNA Polymerase β Subunit (rpoB) Promotes hVISA-to-VISA Phenotypic Conversion of Strain Mu3

ABSTRACT The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N) and graR(N197S) (hereinafter designated graR*). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR*, carrying the mutated two-component response regulator graR*, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR* into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR* thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR* with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR* were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.

Genomic Basis for Methicillin Resistance in Staphylococcus aureus

Since the discovery of the first strain in 1961 in England, MRSA, the most notorious multidrug-resistant hospital pathogen, has spread all over the world. MRSA repeatedly turned down the challenges by number of chemotherapeutics, the fruits of modern organic chemistry. Now, we are in short of effective therapeutic agents against MRSA prevailing among immuno-compromised patients in the hospital. On top of this, we recently became aware of the rise of diverse clones of MRSA, some of which have increased pathogenic potential compared to the classical hospital-associated MRSA, and the others from veterinary sources. They increased rapidly in the community, and started menacing otherwise healthy individuals by causing unexpected acute infection. This review is intended to provide a whole picture of MRSA based on its genetic makeup as a versatile pathogen and our tenacious colonizer.

Stochastic energetics of non-uniform temperature systems

We propose an energetic interpretation of stochastic processes described by Langevin equations with non-uniform temperature. In order to avoid Ito–Stratonovich dilemma, we start with a Kramers equation, and derive a Fokker–Planck equation by the renormalization group method. We give a proper definition of heat for the system. Based on our formulations, we analyze two examples, the Thomson effect and a Brownian motor. The latter realizes the Carnot efficiency.

Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.

Dynamics of a deformable self-propelled domain

We investigate the dynamical coupling between the motion and the deformation of a single self-propelled domain based on two different model systems in two dimensions. One is represented by the set of ordinary differential equations for the center of gravity and two tensor variables characterizing deformations. The other is an active cell model which has an internal mechanism of motility and is represented by the partial differential equation for deformations. Numerical simulations show a rich variety of dynamics, some of which are common to the two model systems. Possible experimental realization of the predictions is discussed.

Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

ABSTRACT Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10−6 or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

Contribution of the Mannan Backbone of Cryptococcal Glucuronoxylomannan and a Glycolytic Enzyme of Staphylococcus aureus to Contact-Mediated Killing of Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy. S. aureus attached to the capsule of C. neoformans, and the ultrastructure of the attached C. neoformans cells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO(4) or protease. Treatment of C. neoformans with NaIO(4) promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowed S. aureus to recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment of S. aureus with protease decreased adherence, suggesting that protein contributed to attachment in S. aureus. In confirmation, side chain-cleaved polysaccharide or defined alpha-(1-->3)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. alpha-(1-->3)-Mannooligosaccharides with a degree of polymerization (DP) of >/=3 induced cluster formation of S. aureus in a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides with a DP of > or =3. Conformations of alpha-(1-->3) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was triosephosphate isomerase (TPI). The enzymatic activities were confirmed by using whole cells of S. aureus. TPI point mutants of S. aureus decreased the ability to interact with C. neoformans. Thus, TPI on S. aureus adheres to the capsule of C. neoformans by recognizing the structure of mannotriose units in the backbone of GXM; we suggest that this contact is required for killing of C. neoformans.