渡邊 真弥

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.

Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes.

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE-B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two-component system, which negatively regulates many virulence factor genes, resulting in a hyper-virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.

Background. Streptococcus dysgalactiae subsp. equisimilis (SDSE) has Lancefield group G or C antigens. Recent epidemiological studies reveal that invasive SDSE infections have been increasing in Asia, Europe, and the United States. The mechanisms and key virulence factors by which SDSE causes invasive diseases are poorly understood.Methods. We analyzed the SDSE transcriptome in vivo during intraperitoneal infection in mice. We also compared the abundance of streptolysin S (SLS) and streptolysin O (SLO) production between clinically dominant stG6792 strains and other clinical isolates.Results. Microarray data suggest that SDSE degraded host tissue polysaccharides by secreting poly/oligosaccharide lyases and simultaneously used the Entner-Doudoroff pathway to metabolize acquired carbohydrates. A global negative virulence gene regulator CsrRS of SDSE modulated the expression of genes encoding SLS and enzymes that metabolize carbohydrates. Moreover, a csrS-deficient mutant induced severe systemic hemolysis in mice. The most frequently isolated stG6792 strains secreted abundant SLS and SLO rather than other SDSE emm types, indicating the potential relationship between production of SLS and SLO and poor outcomes.Conclusions. Our findings suggest that the concomitant regulation of virulence factors that destroy host tissues and metabolic enzymes might play an important role in invasive diseases induced by SDSE. © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging human pathogen that causes life-threatening invasive infections such as streptococcal toxic shock syndrome. Recent epidemiological studies reveal that invasive SDSE infections have been increasing in Asia, Europe, and the United States. Almost all SDSE carry Lancefield group G or C antigen. We have determined the complete genome sequence of a human group C SDSE 167 strain. A comparison of its sequence with that of four SDSE strains, three in Lancefield group G and one in Lancefield group A, showed approximately 90% coverage. Most regions showing little or no homology were located in the prophages. There was no evidence of massive rearrangement in the genome of SDSE 167. Bayesian phylogeny using entire genome sequences showed that the most recent common ancestor of the five SDSE strains appeared 446 years ago. Interestingly, we found that SDSE 167 harbors sugar metabolizing enzymes in a unique region and streptodornase in the phage region, which presumably contribute to the degradation of host tissues and the prompted covRS mutation, respectively. A comparison of these five SDSE strains, which differ in Lancefield group antigens, revealed a gene cluster presumably responsible for the synthesis of the antigenic determinant. These results may provide the basis for molecular epidemiological research of SDSE.

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD(+). This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO(2) incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope ((13)C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from (13)C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.

Background: The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. Methodology/Principal Findings: We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. Conclusion: The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome.

Objectives: The aim of the study was to test in vitro activities of the novel des-F(6)-quinolone DX-619 against methicillin-resistant staphylococci (MRS) isolated in hospitals and communities and to compare its activity with other quinolones, sitafloxacin and levofloxacin, and antibiotics used for the treatment of methicillin-resistant Staphylococcus aureus infection, including vancomycin, teicoplanin, arbekacin, linezolid and quinupristin/dalfopristin. Methods: MICs were determined by the agar dilution method using healthcare-associated MRS (S. aureus including strains with reduced susceptibility to vancomycin, 103; coagulase-negative staphylococci, 87) and community-associated MRS (S. aureus including non-multiresistant oxacillin-resistant strains, 37; coagulase-negative staphylococci, 92). The quinolone resistance-determining regions of gyrA, gyrB, grlA and grlB genes from six strains with reduced susceptibility to DX-619 were sequenced. Results: DX-619 showed the lowest MIC90 values for all categories of strains tested, irrespective of the degree of glycopeptide resistance. The six strains with MIC values of >128 mg/L of levofloxacin commonly carried two mutations in gyrA and two mutations in grlA. DX-619 showed potent activity against strains with MIC values of 2 mg/L. Conclusions: DX-619 was potent against all MRS tested, suggesting that it would be a promising candidate for the treatment of methicillin-resistant S. aureus infection if sufficient in vivo concentrations were safely attained.

Large-scale chromosomal inversions (455 to 535 kbp) or deletions (266 to 320 kbp) were found to accompany spontaneous loss of beta-lactam resistance during drug-free passage of the multiresistant Staphylococcus haemolyticus clinical strain JCSC1435. Identification and sequencing of the rearranged chromosomal loci revealed that ISSha1 of S. haemolyticus is responsible for the chromosome rearrangements.

Staphylocoagulase detection is the hallmark of a Staphylococcus aureus infection. Ten different serotypes of staphylocoagulases have been reported to date. We determined the nucleotide sequences of seven staphylocoagulase genes (coa) and their surrounding regions to compare structures of all 10 staphylocoagulase serotypes, and we inferred their derivations. We found that all staphylocoagulases are comprised of six regions: signal sequence, D1 region, D2 region, central region, repeat region, and C-terminal sequence. Amino acids at both ends, 33 amino acids in the N terminal (the signal sequences and the seven N-terminal amino acids in the D1 region) and 5 amino acids in the C terminal, were exactly identical among the 10 serotypes. The central regions were conserved with identities between 80.6 and 94.1% and similarities between 82.8 and 94.6%. Repeat regions comprising tandem repeats of 27 amino acids with a 92% identity on average were polymorphic in the number of repeats. On the other hand, DI regions other than the seven N-terminal amino acids and D2 regions were less homologous, with diverged identities from 41.5 to 84.5% and 47.0 to 88.9%, respectively, and similarities from 53.5 to 88.7% and 56.8 to 91.9%, respectively, although the predicted prothrombin-binding sites were conserved among them. In contrast, flanking regions of coa were highly homologous, with nucleotide identities of more than 97.1%. Phylogenetic relations among coa did not correlate with those among the flanking regions or housekeeping genes used for multilocus sequence typing. These data indicate that coa could be transmitted to S. aureus, while the less homologous regions in coa presumed to be responsible for different antigenicities might have evolved independently.