Nozomu Hanaoka

Tandem affinity purification of the Candida albicans septin protein complex

A novel vector was constructed to enable the integrative marking of individual genes and the affinity purification of interacting molecules within protein complexes from Candida albicans using a tandem 6×histidine and FLAG epitope tag. The system was verified by purifying the C. albicans septin complex (a self‐associating complex of cytoskeletal proteins) from both yeast and hyphal cells. One‐step affinity purification was insufficient for purification of the protein complex, whereas tandem affinity purification (TAP) gave an extensively purified protein complex with a very low background. Electrophoretic and mass spectrometry analysis showed that the affinity‐purified C. albicans septin complex, which comprises predominantly CaCdc3p, CaCdc10p, CaCdc11p, CaCdc12p and CaSep7p, was not affected by cell morphology. The purified septin complex appeared to have a stoichiometry of 2 CaCdc3p, 1–2 CaCdc10p, 1 CaCdc11p, 2 CaCdc12p and ≤1 CaSep7p. The successful application of TAP to the purification and analysis of the C. albicans septin complex indicates that this technology will have much wider application to proteomic studies of this pathogenic fungus. Copyright

Candida albicans protein kinase CaHsl1p regulates cell elongation and virulence

Saccharomyces cerevisiae Hsl1p is a Ser/Thr protein kinase that regulates cell morphology. We identified Candida albicans CaHSL1 and analysed its function in C. albicans. Cells lacking CaHsl1p exhibited filamentous growth under yeast growth conditions with the filaments elongating more quickly than did those of the wild type under hyphal growth conditions, suggesting that it plays a role in the suppression of cell elongation. Green fluorescent protein‐tagged CaHsl1p colocalized with a septin complex to the bud neck during yeast growth or to a potent septation site during hyphal growth, as expected from the localization in S. cerevisiae. However, the localization of the septin complex did not change in ΔCahsl1, suggesting that CaHsl1p does not participate in septin organization. CaHsl1p was expressed in a cell cycle‐dependent manner and, except for the G1 phase, phosphorylated throughout the cell cycle. In ΔCahsl1 cells, the phosphorylation of a possible CaHsl1p target CaSwe1p decreased, while that of CaCdc28p at tyrosine18 increased. Either an extra copy of the tyrosine18‐mutated CaCdc28p or deletion of CaSWE1 suppressed the cell elongation phenotype caused by CaHSL1 deletion. Furthermore, ΔCahsl1 exhibited reduced virulence in the mouse systemic candidiasis model. Thus, the CaHsl1p‐CaSwe1p‐CaCdc28p pathway appears important in the cell elongation of both the yeast and hyphal forms and to the virulence of C. albicans.

Human Rickettsia heilongjiangensis infection, Japan.

A case of Rickettsia heilongjiangensis infection in Japan was identified in a 35-year-old man who had rash, fever, and eschars. Serum contained R. heilongjiangensis antibodies, and eschars contained R. heilongjiangensis DNA. R. heilongjiangensis was also isolated from ticks in the suspected geographic area of infection.

Identification of the Putative Protein Phosphatase Gene PTC1 as a Virulence-Related Gene Using a Silkworm Model of Candida albicans Infection

ABSTRACT Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.

Surveillance of Adenovirus D in patients with epidemic keratoconjunctivitis from Fukui Prefecture, Japan, 1995–2010

Human adenoviruses species D (HAdV‐D) are known to cause severe epidemic keratoconjunctivitis. However, the isolation rate of HAdV‐D is not high, because HAdV‐D is usually slow to propagate. Although new types of HAdV‐D have been reported, accurate surveillance has not been performed because of difficulties in culturing the viruses and lack of a practical identification method. In this study, HAdV‐Ds were detected and identified from patients with epidemic keratoconjunctivitis in the Fukui Prefecture during 1995–2010 by PCR, loop‐mediated isothermal amplification (LAMP) of DNA, and conventional virus isolation and neutralization tests. All samples were subjected to culture and PCR and LAMP. A total of 124 strains of HAdV‐D were detected from 157 patients with epidemic keratoconjunctivitis. The strains consisted of the following types: D8 (n = 8), D19 (n = 4), D37 (n = 40), D53 (n = 5), D54 (n = 66), and D56 (n = 1). Among these, D53, D54, and D56 are new types that have been reported recently. The results of this study demonstrated that new types of HAdV‐D caused epidemic keratoconjunctivitis during 1995–2010, and included an outbreak of keratoconjunctivitis caused by HAdV‐D54. The LAMP method was able to detect and identify HAdV‐D53 and HAdV–D54 in 1 hr, and may therefore be applicable for use at the bedside. J. Med. Virol. 84:81–86, 2011.

Multilocus Sequence Typing Implicates Rodents as the Main Reservoir Host of Human-Pathogenic Borrelia garinii in Japan

ABSTRACT Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.

Diagnostic Assay for Rickettsia japonica

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains.

Male non-gonococcal urethritis: From microbiological etiologies to demographic and clinical features.

To detect microorganisms responsible for male acute urethritis and to define the microbiology of non‐gonococcal urethritis.

First isolation of a new type of human adenovirus (genotype 79), species Human mastadenovirus B (B2) from sewage water in Japan

Human mastadenoviruses (HAdVs) are highly infectious viral pathogens that survive for prolonged periods in environmental waters. We monitored the presence of HAdVs in sewage waters between April 2014 and March 2015. A total of 27 adenoviral strains were detected in 75% (18/24 in occasion‐base) of 24 wastewater collected samples. We identified the types of the strains as HAdV‐C2 (n = 5), HAdV‐A31 (5), HAdV‐C1 (4), HAdV‐B3 (4), HAdV‐C5 (4), HAdV‐B11 (2), P11H34F11 (2), and HAdV‐D56 (1). The complete genome sequence of one P11H34F11 (strain T150125) was determined by next‐generation sequencing and compared to other genome sequences of HAdV‐B strains. The comparisons revealed evidence of a recombination event with breaking point in the hexon encoding region, which evidenced high similarity to HAdV‐B34, while half of the rest of the genome showed similarity to HAdV‐B11, including regions encoding fiber and E3 region proteins. The penton base encoding region seemed to be a recombinant product of HAdV‐B14, ‐34; however, it was evidenced to be divergent to both as a novel type despite showing low bootstrap to support a new clade. We propose T150125 (P11H34F11) is a strain of a novel genotype, HAdV‐79. These results support the usefulness of environmental surveillance approaches to monitor circulating HAdVs including novel types.

Characterization of genome sequences and clinical features of coxsackievirus A6 strains collected in Hyogo, Japan in 1999-2013.

Coxsackievirus A6 (CV‐A6) is an enterovirus, which is known to cause herpangina. However, since 2009 it has frequently been isolated from children with hand, foot, and mouth disease (HFMD). In Japan, CV‐A6 has been linked to HFMD outbreaks in 2011 and 2013. In this study, the full‐length genome sequencing of CV‐A6 strains were analyzed to identify the association with clinical manifestations. Five thousand six hundred and twelve children with suspected enterovirus infection (0‐17 years old) between 1999 and 2013 in Hyogo Prefecture, Japan, were enrolled. Enterovirus infection was confirmed with reverse transcriptase‐PCR in 753 children (791 samples), 127 of whom (133 samples) were positive for CV‐A6 based on the direct sequencing of the VP4 region. The complete genomes of CV‐A6 from 22 positive patients with different clinical manifestations were investigated. A phylogenetic analysis divided these 22 strains into two clusters based on the VP1 region; cluster I contained strains collected in 1999‐2009 and mostly related to herpangina, and cluster II contained strains collected in 2011‐2013 and related to HFMD outbreak. Based on the full‐length polyprotein analysis, the amino acid differences between the strains in cluster I and II were 97.7 ± 0.28%. Amino acid differences were detected in 17 positions within the polyprotein. Strains collected in 1999‐2009 and those in 2011‐2013 were separately clustered by phylogenetic analysis based on 5′UTR and 3Dpol region, as well as VP1 region. In conclusion, HFMD outbreaks by CV‐A6 were recently frequent in Japan and the accumulation of genomic change might be associated with the clinical course.