A novel Zn2-Cys6 transcription factor clcA contributes to copper homeostasis in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is the most important pathogenic fungus among Aspergillus species associated with aspergillosis. A. fumigatus is exposed to diverse environmental stresses in the hosts during infection such as an excess of essential metal copper. To gain further insights into copper homeostasis, we generated an A. fumigatus laboratory evolved strain with increased fitness in copper stress, and identified the mutation in a Zn2-Cys6 type transcription factor clcA. We examined the role of clcA using the evolved and ∆clcA strains. The ∆clcA strain exhibited defective growth on minimal medium, PDA and copper-repleted medium, and defective conidiogenesis and conidial pigmentation. We found that clcA was required for the expressions of genes involved in conidiogenesis, conidial pigmentation, and transporters cdr1B and mfsB related to azole resistance. clcA was dispensable for the virulence in silkworm infection model. We report here that clcA plays an important role in hyphal growth, conidiogenesis, and copper adaptation.

Dysfunction of Ras-GAP protein AfgapA contributes to hypoxia fitness in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is the most important pathogenic fungus among Aspergillus species associated with aspergillosis. A. fumigatus must adapt to hypoxic microenvironments to survive and thrive in human lungs. To gain further insights into hypoxic adaptation, we generated a laboratory-evolved strain (Afs35-G20) harboring hypoxia fitness, and identified a nonsense mutation in AfgapA encoding a Ras-GAP protein, which could result in the deletion of 22 amino acids at the C-terminus. We investigated the role of AfgapA in hypoxia fitness by constructing Afs35-G20-AfgapAWT, and ∆AfgapA. Indeed, the hypoxia fitness of Afs35-G20 was reversed by introducing AfgapAWT. ∆AfgapA exhibited greater hypoxia fitness and hypervirulence in the silkworm infection model, indicating that AfgapA is responsible for hypoxia fitness, particularly in liquid cultures. Taken together, the AfgapA dysfunction may lead to the downregulation of its Ras substrate(s), reflecting several phenotypes such as increased hypoxia fitness, hypervirulence, poor conidiation, and conidial pigmentation. Here, we report the function of a Ras-GAP protein AfgapA in A. fumigatus for the first time.

Cutaneous Immunoprofiles of Three Spotted Fever Group Rickettsia Cases.

Spotted fever group rickettsia (SFGR) can cause mild to fatal illness. The early interaction between the host and rickettsia in skin is largely unknown, and the pathogenesis of severe rickettsiosis remains an important topic. A surveillance of SFGR infection by PCR of blood and skin biopsy specimens followed by sequencing and immunohistochemical (IHC) detection was performed on patients with a recent tick bite between 2013 and 2016. Humoral and cutaneous immunoprofiles were evaluated in different SFGR cases by serum cytokine and chemokine detection, skin IHC staining, and transcriptome sequencing (RNA-seq). A total of 111 SFGR cases were identified, including 79 "Candidatus Rickettsia tarasevichiae," 22 Rickettsia raoultii, 8 Rickettsia sibirica, and 2 Rickettsia heilongjiangensis cases. The sensitivity to detect SFGR in skin biopsy specimens (9/24, 37.5%) was significantly higher than that in blood samples (105/2,671, 3.9%) (P < 0.05). As early as 1 day after the tick bite, rickettsiae could be detected in the skin. R. sibirica infection was more severe than "Ca Rickettsia" and R. raoultii infections. Increased levels of serum interleukin-18 (IL-18), IP10, and monokine induced by gamma interferon (MIG) and decreased levels of IL-2 were observed in febrile patients infected with R. sibirica compared to those infected with "Ca Rickettsia." RNA-seq and IHC staining could not discriminate between SFGR-infected and uninfected tick bite skin lesions. However, the type I interferon (IFN) response was differently expressed between R. sibirica and R. raoultii infections at the cutaneous interface. It is concluded that skin biopsy specimens were more reliable for the detection of SFGR infection in human patients although the immunoprofile may be complicated by immunomodulators induced by the tick bite.

Genomic diversity of the pathogenic fungus Aspergillus fumigatus in Japan reveals the complex genomic basis of azole resistance.

Aspergillus fumigatus is a pathogenic fungus with a global distribution. The emergence of azole-resistant A. fumigatus (ARAf) other than the TR-mutants is a problem in Japan. Additionally, the genetic diversity of A. fumigatus strains in Japan remains relatively unknown. Here we show the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. First, we analyzed the genome sequences of 171 strains from Japan as well as the antifungal susceptibility of these strains. Next, we conducted a population analysis of 876 strains by combining the available genomic data for strains isolated worldwide, which were grouped in six clusters. Finally, a genome-wide association study identified the genomic loci associated with ARAf strains, but not the TR-mutants. These results highlight the complexity of the genomic mechanism underlying the emergence of ARAf strains other than the TR-mutants.

Skin infectome of patients with a tick bite history.

Introduction: Ticks are the most important obligate blood-feeding vectors of human pathogens. With the advance of high-throughput sequencing, more and more bacterial community and virome in tick has been reported, which seems to pose a great threat to people. Methods: A total of 14 skin specimens collected from tick-bite patients with mild to severe symptoms were analyzed through meta-transcriptomic sequencings. Results: Four bacteria genera were both detected in the skins and ticks, including Pseudomonas, Acinetobacter, Corynebacterium and Propionibacterium, and three tick-associated viruses, Jingmen tick virus (JMTV), Bole tick virus 4 (BLTV4) and Deer tick mononegavirales-like virus (DTMV) were identified in the skin samples. Except of known pathogens such as pathogenic rickettsia, Coxiella burnetii and JMTV, we suggest Roseomonas cervicalis and BLTV4 as potential new agents amplified in the skins and then disseminated into the blood. As early as 1 day after a tick-bite, these pathogens can transmit to skins and at most four ones can co-infect in skins. Discussion: Advances in sequencing technologies have revealed that the diversity of tick microbiome and virome goes far beyond our previous understanding. This report not only identifies three new potential pathogens in humans but also shows that the skin barrier is vital in preventing horizontal transmissions of tick-associated bacteria or virus communities to the host. It is the first research on patients' skin infectome after a tick bite and demonstrates that more attention should be paid to the cutaneous response to prevent tick-borne illness.

Virome and Blood Meal-Associated Host Responses in Ixodes persulcatus Naturally Fed on Patients.

The long-lasting co-evolution of ticks with pathogens results in mutual adaptation. Blood-feeding is one of the critical physiological behaviors that have been associated with the tick microbiome; however, most knowledge was gained through the study of laboratory-reared ticks. Here we detached Ixodes persulcatus ticks at different stages of blood-feeding from human patients and performed high-throughput transcriptomic analysis on them to identify their virome and genes differentially expressed between flat and fully fed ticks. We also traced bloodmeal sources of those ticks and identified bats and three other potential mammalian hosts, highlighting the public health significance. We found Jingmen tick virus and 13 putative new viruses belonging to 11 viral families, three of which even exhibited high genetic divergence from viruses previously reported in the same tick species from the same geographic region. Furthermore, differential expression analysis suggested a downregulation of antioxidant genes in the fully fed I. persulcatus ticks, which might be related to bloodmeal-related redox homeostasis. Our work highlights the significance of active surveillance of tick viromes and suggests a role of reactive oxygen species (ROS) in modulating changes in the microbiome during blood-feeding.

Human Case Infected With Babesia venatorum: A 5-Year Follow-Up Study.

BACKGROUND: Human babesiosis is a common zoonosis caused by Babesia and is attracting an increasing concern worldwide. The natural course of babesiosis infection and how the human immune system changes during the course of babesiosis infection are not clear. METHODS: We followed up 1 case infected with Babesia venatorum for 5 years. The patient was immune-intact and received no standard treatment. Clinical data were obtained from medical records. Microbiological tests, ribonucleic acid (RNA) sequence, and serum cytokines and chemokines were detected at different time points. RESULTS: The patient was confirmed as B venatorum infection based on his tick-bite history, clinical manifestations, and positive results of microbiological tests. The parasitemia of the patient persisted for approximately 2 months. With flu-like symptoms aggravating, most cytokines and chemokines in RNA and protein levels increased progressively and reached the peak when fever occurred; and their concentrations decreased to baseline during the same time as clearance of babesia parasites. CONCLUSIONS: Babesia venatorum infection could take a mild self-limited course in immune-intact individuals. The natural changes of most cytokines and chemokines demonstrated very similar trends, which correlated with blood parasitemia and clinical manifestations. Cytokine profiles involving multiple inflammatory cytokines might be a good indicator of babesia infection.

Draft Genome Sequence of Aspergillus awamori IFM 58123NT.

Species of the Aspergillus section Nigri are taxonomically very complex. The taxonomic assignment of Aspergillus awamori is unclear. Here, we present the draft genome sequence of A. awamori strain IFM 58123NT.

[Effect of BSA on random amplified polymorphic DNA (RAPD) in plants].

Using Metasequoia glyptostroboides and Heptacodium miconioides DNA as templates,the effect of bovine serum albumin (BSA) on RAPD in plants was studied. The results showed that suitable concentrations of BSA used in Metasequoia glyptostroboides and Heptacodium miconioides RAPD were different, which were 0.6 microg/microl and 1 microg/microl, respectively. The inhibition of acetylated BSA on the amplification of plant RAPD could be relieved by BSA. BSA could reduce the dosage of Taq DNA polymerase.

Elevated urinary levels of cystatin C and neutrophil gelatinase-associated lipocalin in Henoch-Schönlein purpura patients with renal involvement.

Henoch-Schönlein purpura (HSP) is a commonest systemic vasculitis in childhood. The long-term prognosis of HSP is determined by the degree of renal involvement. The aim of this study is to search novel clinically applicable biomarkers to evaluate renal involvement in HSP patients. 20 bio-indexes in urine samples were simultaneously screened by antibody array assay. We indicated that urinary levels of cystatin C (Cys C) and neutrophil gelatinase-associated lipocalin (NGAL) in HSP patients with renal involvement were significantly higher than those without renal involvement and healthy controls. Furthermore, ELISA was used to analyze urinary Cys C and NGAL levels in HSP patients with or without renal involvement, atopic dermatitis (AD) patients and healthy controls. Our results demonstrated that urinary Cys C and NGAL levels in HSP patients with renal involvement were significantly elevated, when compared with those without renal involvement, AD patients and control subjects. In addition, by receiver operating characteristic (ROC) curve analysis, we demonstrated that the area under the ROC curve of NGAL (0.789) was larger than that of Cys C (0.692). Taken together, we show firstly that urinary Cys C and NGAL levels is abnormally elevated in HSP patients with renal involvement. We suggest that urinary Cys C and NGAL are novel useful biomarkers of renal involvement in HSP patients.

Elevation of matrix metalloproteinase-9 level in cerebrospinal fluid of tick-borne encephalitis patients is associated with IgG extravassation and disease severity.

BACKGROUND: Tick-borne encephalitis (TBE), caused by tick-borne encephalitis virus (TBEV), is an infectious disease involving the central nervous system (CNS). The pathogenesis of CNS injury has not been clearly demonstrated. Matrix metalloproteinase-9 (MMP-9) and some cytokines, such as interleukin 6 (IL-6), may play important roles in the disruption of the blood-brain barrier (BBB) and the pathogenesis of TBE. METHODS: 72 cerebrospinal fluid (CSF) samples were collected from TBE patients in north eastern China. IgG levels in CSF and serum were compared and MMP-9 and IL-6 levels were evaluated by ELISA. The correlation between the elevated MMP-9 levels and IgG extravasation, disease severity, and neuroinflammation was analyzed. RESULTS: Increased concentration of MMP-9 was detected in some of the CSF samples, and the elevation was found to be closely related to CSF TBEV IgG extravasation and enhancement of IL-6 expression. Moreover, elevated levels of MMP-9 were found to be correlated with IL-6 enhancement. Four of the 72 patients, the ones who died, presented with high CSF MMP-9 levels. CONCLUSIONS: In TBE patients, elevated CSF MMP-9 levels were associated with brain inflammatory reaction, disruption of the blood-brain barrier, and disease severity.