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Candida auris is an invasive and multidrug-resistant ascomycetous yeast that is under global surveillance. All clinical cases of C. auris infection diagnosed from 1997 to 2019 in Japan were non-invasive and sporadic otitis media cases. In the present study, we performed whole-genome sequencing of seven C. auris strains isolated from patients with otitis media in Japan, all of which belonged to clade II. Comparative genome analysis using the high-quality draft genome sequences JCM 15448T revealed that single nucleotide variations (SNVs), clade-specific accessory genes, and copy number variations (CNVs) were identified in each C. auris clade. A total of 61 genes involved in cell wall and stress response-related functions was absent in clade II, and the pattern of conserved CNVs in each clade was more stable in clade II than in other clades. Our data suggest that the genomic structural diversity is stable in C. auris isolated from each biogeographic location, and Japanese strains isolated from patients with otitis media might belong to an ancestral type of C. auris. One Japanese strain, TWCC 58362, with reduced susceptibility to fluconazole, exhibited no mutation in ergosterol biosynthesis-related genes (ERG). However, TWCC 58362-specific variations, including SNVs, indels, and CNVs were detected, suggesting that gene duplication events in C. auris might contribute to antifungal drug resistance. Taken together, we demonstrated that genomic structural variations in C. auris could correlate to geographical dissemination, epidemiology, lesions in the host, and antifungal resistance. [ABSTRACT FROM AUTHOR]

The regulation of paramyxovirus RNA synthesis by host proteins is poorly understood. Here, we identified a novel regulation mechanism of paramyxovirus RNA synthesis by the Hsp90 co-chaperone R2TP complex. We showed that the R2TP complex interacted with the paramyxovirus polymerase L protein and that silencing of the R2TP complex led to uncontrolled upregulation of mumps virus (MuV) gene transcription but not genome replication. Regulation by the R2TP complex was critical for MuV replication and evasion of host innate immune responses. The R2TP complex also regulated measles virus (MeV) RNA synthesis, but its function was inhibitory and not beneficial to MeV, as MeV evaded host innate immune responses in the absence of the R2TP complex. The identification of the R2TP complex as a critical host factor sheds new light on the regulation of paramyxovirus RNA synthesis. [ABSTRACT FROM AUTHOR]

Objectives: Automated online software tools that analyse whole genome sequencing (WGS) data without the need for bioinformatics expertise can motivate the implementation of WGS-based molecular drug susceptibility testing (DST) in routine diagnostic settings for tuberculosis (TB). Pyrazinamide (PZA) is a key drug for current and future TB treatment regimens; however, it was reported that predictive power for PZA resistance by the available tools is low. Therefore, this low predictive power may make users hesitant to use the tools. This study aimed to elucidate why and to uncover the real performance of the tools when taking into account their variation calling lists (manual inspection), not just their automated reporting system (default setting) that was evaluated by previous studies. Methods: WGS data from 191 datasets comprising 108 PZA-resistant and 83 susceptible strains were used to evaluate the potential performance of the available online tools (TB Profiler, TGS-TB, PhyResSE, and CASTB) for predicting phenotypic PZA resistance. Results: When taking into consideration the variation calling lists, 73 variants in total (47 non-synonymous mutations and 26 indels) in pncA were detected by TGS-TB and PhyResSE, covering all mutations for the 108 PZA-resistant strains. The 73 variants were confirmed by Sanger sequencing. TB Profiler also detected all but three complete loss, two large deletion at the 3’-end, and one relatively large insertion of pncA. On the other hand, many of the 73 variants were lacking in the automated reporting systems except by TGS-TB; of these variants, CASTB detected only 20. By applying the ‘non-wild type sequence’ approach for predicting PZA resistance, accuracy of the results significantly improved compared with that of the automated results obtained by each tool. Conclusion: Users can obtain more accurate predictions for PZA resistance than previously reported by manually checking the results and applying the ‘non-wild type sequence’ approach. [ABSTRACT FROM AUTHOR]

Carbapenemase-producing Enterobacteriaceae (CPE) are a global concern because these bacteria are resistant to almost all β-lactams. Horizontal interspecies gene transfer via plasmid conjugation has increased the global dissemination of CPE. Recently, an Enterobacteriaceae strain positive for carbapenemase gene but showing a carbapenem-susceptible phenotype was identified, suggesting that these susceptible strains may be challenging to detect solely via antimicrobial susceptibility tests without molecular analysis. Here, we isolated a blaIMP-6 carbapenemase-gene positive but imipenem- and meropenem-susceptible Escherichia coli (ISMS-E) strain A56-1S (imipenem and meropenem minimum inhibitory concentration, ≤ 0.125 mg/L), from a human urine specimen in Japan. A56-1S was carbapenemase negative by the Carba NP test, suggesting that IMP-6 production was low or undetectable. Thus, to characterize the mechanism of this phenotype, a meropenem-resistant E. coli A56-1R strain was obtained using meropenem-selection. A56-1R was positive for carbapenemase production by the Carba NP test, and blaIMP-6 transcription in A56-1R was 53-fold higher than in A56-1S, indicating that blaIMP-6 in A56-1S is negatively regulated at the transcriptional level. Comparative genomic analysis between the two strains revealed that the alleviation of restriction of DNA (ardK) gene encoding a putative transcription factor is disrupted by the IS26 insertion in A56-1R. A cotransformation assay of ardK and the regulatory element upstream of blaIMP-6 showed repression of blaIMP-6 transcription, indicating that ArdK negatively modulates blaIMP-6 transcription. ArdK binding and affinity assays demonstrated that ArdK directly binds to the regulatory element upstream of blaIMP-6 with dissociation constant values comparable to those of general transcription factors. The IMP-6 carbapenemase showed low hydrolytic activity against imipenem, resulting in an imipenem-susceptible and meropenem-resistant (ISMR) phenotype (previously reported as a stealth phenotype). However, the low expression of IMP-6 in the A56-1S strain could be a typical characteristic of ISMS-E due to gene repression, indicating that conventional antimicrobial susceptibility tests might be unable to detect such strains even when using both imipenem and meropenem. Bacteria that exhibit the ISMS phenotype could play a potential role as undetectable reservoirs and might facilitate gene transfer to other organisms while avoiding detection. [ABSTRACT FROM AUTHOR]

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-nduced ene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment. [ABSTRACT FROM AUTHOR]

Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn’s disease and ulcerative colitis (UC). Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb) isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb) and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS) and 13 Fusobacterium adhesion (FadA) paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium. [ABSTRACT FROM AUTHOR]

It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs) have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35) is found in the little brown bat (Myotis lucifugus) genome. Putative mlEFL35-derived protein (mlEFL35p) contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN) production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-β promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor. [ABSTRACT FROM AUTHOR]

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for elderly adults. Until now, nearly all GBS with reduced penicillin susceptibility (PRGBS) have shown β-hemolytic activity and grow on sheep blood agar. However, we have previously reported three PRGBS clinical isolates harboring a CylK deletion that form small less hemolytic colonies. In this study, we examined the causes of small, less hemolytic colony formation in these clinical isolates. Isogenic strains were sequenced to identify the mutation related to a small colony size. We identified a 276_277insG nucleic acid insertion in the thiamin pyrophosphokinase (tpk) gene, resulting in premature termination at amino acid 103 in TPK, as a candidate mutation responsible for small colony formation. The recombinant strain Δtpk, which harbored the 276_277insG insertion in the tpk gene, showed small colony formation. The recombinant strain ΔcylK, which harbored the G379T substitution in cylK, showed a reduction in hemolytic activity. The phenotypes of both recombinant strains were complemented by the expression of intact TPK or CylK, respectively. Moreover, the use of Rapid ID 32 API and VITEK MS to identify strains as GBS was evaluated clinical isolates and recombinant strains. VITEK MS, but not Rapid ID 32 API, was able to accurately identify the strains as GBS. In conclusion, we determined that mutations in tpk and cylK caused small colonies and reduced hemolytic activity, respectively, and characterized the clinical isolates in detail. [ABSTRACT FROM AUTHOR]

Background/Aim: In the colorectum, lymphoid follicles hyperplasia (LH) is sometimes observed as small, round, yellowish-white nodules. The novel image-enhanced endoscopy system named blue laser imaging (BLI) provides enhanced the contrast of surface vessels using lasers for light illumination. We investigated the endoscopic features of LH observed by using BLI endoscopy and its association with chronic bowel symptoms. Patients/Methods: 300 participants undergoing colonoscopy for various indications were enrolled. Entire colorectum was observed by using BLI-bright mode with non-magnification view. LH was defined as well demarcated white nodules. Elevated LH with erythema was distinguished as LH severe. Results: LHs were observed more clearly by using BLI-bright mode compared to conventional white light colonoscopy and were also histologically confirmed as intense infiltration of lymphocytes or plasmacytes. LH was observed in 134 subjects (44.6%) and 67 (22.3%) were LH severe. LH was associated younger age (Odds ratio (OR) = 1.05, 95%Confidence Interval (95%CI) = 1.03–1.07, P<0.0001) and chronic bowel symptoms including constipation, hard stools, diarrhea and loose stools (all LH: OR = 4.03, 95%CI = 2.36–6.89, P<0.0001, LH severe: OR = 5.31, 95%CI = 2.64–10.71, P<0.0001). LH severe was closely associated with both constipation associated symptoms (OR = 3.94, 95%CI = 1.79–8.66, P = 0.0007) and diarrhea associated symptoms (OR = 5.22, 95%CI = 2.09–13.05, P = 0.0004). In particular, LH severe in the ascending colon was strongly associated with bowel symptoms (P<0.0001). Conclusion: LH, visualized by using BLI endoscopy was associated with bowel symptom, raising the possibility of pathogenic role of this endoscopic finding in the functional lower gastrointestinal disorders. [ABSTRACT FROM AUTHOR]

Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years. [ABSTRACT FROM AUTHOR]

Antibody-dependent enhancement (ADE) of Ebola virus (EBOV) infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa)-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs) is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection. [ABSTRACT FROM AUTHOR]

Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein—Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus group subsp., such as M. massiliense, M. abscessus sensu stricto and M. bolletii, are an environmental organism found in soil, water and other ecological niches, and have been isolated from respiratory tract infection, skin and soft tissue infection, postoperative infection of cosmetic surgery. To determine the unique genetic feature of M. massiliense, we sequenced the complete genome of M. massiliense type strain JCM 15300 (corresponding to CCUG 48898). Comparative genomic analysis was performed among Mycobacterium spp. and among M. abscessus group subspp., showing that additional ß-oxidation-related genes and, notably, the mammalian cell entry (mce) operon were located on a genomic island, M. massiliense Genomic Island 1 (MmGI-1), in M. massiliense. In addition, putative anaerobic respiration system-related genes and additional mycolic acid cyclopropane synthetase-related genes were found uniquely in M. massiliense. Japanese isolates of M. massiliense also frequently possess the MmGI-1 (14/44, approximately 32%) and three unique conserved regions (26/44; approximately 60%, 34/44; approximately 77% and 40/44; approximately 91%), as well as isolates of other countries (Malaysia, France, United Kingdom and United States). The well-conserved genomic island MmGI-1 may play an important role in high growth potential with additional lipid metabolism, extra factors for survival in the environment or synthesis of complex membrane-associated lipids. ORFs on MmGI-1 showed similarities to ORFs of phylogenetically distant M. avium complex (MAC), suggesting that horizontal gene transfer or genetic recombination events might have occurred within MmGI-1 among M. massiliense and MAC. [ABSTRACT FROM AUTHOR]

Previous work has shown that the obligate intracellular amoebal endosymbiont Neochlamydia S13, an environmental chlamydia strain, has an amoebal infection rate of 100%, but does not cause amoebal lysis and lacks transferability to other host amoebae. The underlying mechanism for these observations remains unknown. In this study, we found that the host amoeba could completely evade Legionella infection. The draft genome sequence of Neochlamydia S13 revealed several defects in essential metabolic pathways, as well as unique molecules with leucine-rich repeats (LRRs) and ankyrin domains, responsible for protein-protein interaction. Neochlamydia S13 lacked an intact tricarboxylic acid cycle and had an incomplete respiratory chain. ADP/ATP translocases, ATP-binding cassette transporters, and secretion systems (types II and III) were well conserved, but no type IV secretion system was found. The number of outer membrane proteins (OmcB, PomS, 76-kDa protein, and OmpW) was limited. Interestingly, genes predicting unique proteins with LRRs (30 genes) or ankyrin domains (one gene) were identified. Furthermore, 33 transposases were found, possibly explaining the drastic genome modification. Taken together, the genomic features of Neochlamydia S13 explain the intimate interaction with the host amoeba to compensate for bacterial metabolic defects, and illuminate the role of the endosymbiont in the defense of the host amoebae against Legionella infection. [ABSTRACT FROM AUTHOR]

Previous work has shown that the obligate intracellular amoebal endosymbiont Neochlamydia S13, an environmental chlamydia strain, has an amoebal infection rate of 100%, but does not cause amoebal lysis and lacks transferability to other host amoebae. The underlying mechanism for these observations remains unknown. In this study, we found that the host amoeba could completely evade Legionella infection. The draft genome sequence of Neochlamydia S13 revealed several defects in essential metabolic pathways, as well as unique molecules with leucine-rich repeats (LRRs) and ankyrin domains, responsible for protein-protein interaction. Neochlamydia S13 lacked an intact tricarboxylic acid cycle and had an incomplete respiratory chain. ADP/ATP translocases, ATP-binding cassette transporters, and secretion systems (types II and III) were well conserved, but no type IV secretion system was found. The number of outer membrane proteins (OmcB, PomS, 76-kDa protein, and OmpW) was limited. Interestingly, genes predicting unique proteins with LRRs (30 genes) or ankyrin domains (one gene) were identified. Furthermore, 33 transposases were found, possibly explaining the drastic genome modification. Taken together, the genomic features of Neochlamydia S13 explain the intimate interaction with the host amoeba to compensate for bacterial metabolic defects, and illuminate the role of the endosymbiont in the defense of the host amoebae against Legionella infection. [ABSTRACT FROM AUTHOR]