Glycolipid antigen recognition by invariant natural killer T cells and its role in homeostasis and antimicrobial responses.

Due to the COVID-19 pandemic, the importance of developing effective vaccines has received more attention than ever before. To maximize the effects of vaccines, it is important to select adjuvants that induce strong and rapid innate and acquired immune responses. Invariant natural killer T (iNKT) cells, which constitute a small population among lymphocytes, bypass the innate and acquired immune systems through the rapid production of cytokines after glycolipid recognition; hence, their activation could be used as a vaccine strategy against emerging infectious diseases. Additionally, the diverse functions of iNKT cells, including enhancing antibody production, are becoming more understood in recent years. In this review, we briefly describe the functional subset of iNKT cells and introduce the glycolipid antigens recognized by them. Furthermore, we also introduce novel vaccine development taking advantages of iNKT cell activation against infectious diseases.

Unveiling the role of preceding seasonal influenza in the development of bacteremic pneumococcal pneumonia in older adults before the COVID-19 pandemic in Japan.

OBJECTIVE: We aimed to investigate the impact of preceding seasonal influenza on the clinical characteristics of adult patients with invasive pneumococcal disease (IPD) in Japan. METHODS: Data for 1722 adult patients with IPD were analyzed before (2017-2019) and during the COVID-19 pandemic (2020-2022). RESULTS: The seasonal influenza epidemic disappeared soon after the emergence of the pandemic. Compared with that before the pandemic (66.7%), we observed a lower bacteremic pneumonia proportion in patients with IPD during the pandemic (55.6%). The clinical presentations of IPD cases significantly differed between those with and without preceding influenza. The proportion of bacteremic pneumonia was higher in IPD patients with preceding influenza than in those without in both younger (44.9% vs 84.2%) and older adults (65.5% vs 87.0%) before the pandemic. The case fatality rate was significantly higher in IPD patients with preceding influenza (28.3%) than in those without (15.3%) in older adults before the pandemic (P = 0.020). Male and aging are high risk factors for death in older patients with IPD who had preceding influenza. CONCLUSION: Our study reveals that preceding seasonal influenza plays a role in the development of bacteremic pneumococcal pneumonia, increasing the risk of death in older adults.

IL-27 regulates the differentiation of follicular helper NKT cells via metabolic adaptation of mitochondria.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies.

Differences of white matter structure for diffusion kurtosis imaging using voxel-based morphometry and connectivity analysis.

Objectives: In a clinical study, diffusion kurtosis imaging (DKI) has been used to visualize and distinguish white matter (WM) structures' details. The purpose of our study is to evaluate and compare the diffusion tensor imaging (DTI) and DKI parameter values to obtain WM structure differences of healthy subjects. Methods: Thirteen healthy volunteers (mean age, 25.2 years) were examined in this study. On a 3-T MRI system, diffusion dataset for DKI was acquired using an echo-planner imaging sequence, and T1-weghted (T1w) images were acquired. Imaging analysis was performed using Functional MRI of the brain Software Library (FSL). First, registration analysis was performed using the T1w of each subject to MNI152. Second, DTI (eg, fractional anisotropy [FA] and each diffusivity) and DKI (eg, mean kurtosis [MK], radial kurtosis [RK], and axial kurtosis [AK]) datasets were applied to above computed spline coefficients and affine matrices. Each DTI and DKI parameter value for WM areas was compared. Finally, tract-based spatial statistics (TBSS) analysis was performed using each parameter. Results: The relationship between FA and kurtosis parameters (MK, RK, and AK) for WM areas had a strong positive correlation (FA-MK, R2 = 0.93; FA-RK, R2 = 0.89) and a strong negative correlation (FA-AK, R2 = 0.92). When comparing a TBSS connection, we found that this could be observed more clearly in MK than in RK and FA. Conclusions: WM analysis with DKI enable us to obtain more detailed information for connectivity between nerve structures. Advances in knowledge: Quantitative indices of neurological diseases were determined using segmenting WM regions using voxel-based morphometry processing of DKI images.

Instantaneous Clearing of Biofilm (iCBiofilm): an optical approach to revisit bacterial and fungal biofilm imaging.

Whole-biofilm imaging at single-cell resolution is necessary for system-level analysis of cellular heterogeneity, identification of key matrix component functions and response to immune cells and antimicrobials. To this end, we developed a whole-biofilm clearing and imaging method, termed instantaneous clearing of biofilm (iCBiofilm). iCBiofilm is a simple, rapid, and efficient method involving the immersion of biofilm samples in a refractive index-matching medium, enabling instant whole-biofilm imaging with confocal laser scanning microscopy. We also developed non-fixing iCBiofilm, enabling live and dynamic imaging of biofilm development and actions of antimicrobials. iCBiofilm is applicable for multicolor imaging of fluorescent proteins, immunostained matrix components, and fluorescence labeled cells in biofilms with a thickness of several hundred micrometers. iCBiofilm is scalable from bacterial to fungal biofilms and can be used to observe biofilm-neutrophil interactions. iCBiofilm therefore represents an important advance for examining the dynamics and functions of biofilms and revisiting bacterial and fungal biofilm formation.

Small-Molecule-Induced Activation of Cellular Respiration Inhibits Biofilm Formation and Triggers Metabolic Remodeling in Staphylococcus aureus.

Staphylococcus aureus, a major pathogen of community-acquired and nosocomial-associated infections, forms biofilms consisting of extracellular matrix-embedded cell aggregates. S. aureus biofilm formation on implanted medical devices can cause local and systemic infections due to the dispersion of cells from the biofilms. Usually, conventional antibiotic treatments are not effective against biofilm-related infections, and there is no effective treatment other than removing the contaminated devices. Therefore, the development of new therapeutic agents to combat biofilm-related infections is urgently needed. We conducted high-throughput screening of S. aureus biofilm inhibitors and obtained a small compound, JBD1. JBD1 strongly inhibits biofilm formation of S. aureus, including methicillin-resistant strains. In addition, JBD1 activated the respiratory activity of S. aureus cells and increased the sensitivity to aminoglycosides. Furthermore, it was shown that the metabolic profile of S. aureus was significantly altered in the presence of JBD1 and that metabolic remodeling was induced. Surprisingly, these JBD1-induced phenotypes were blocked by adding an excess amount of the electron carrier menaquinone to suppress respiratory activation. These results indicate that JBD1 induces biofilm inhibition and metabolic remodeling through respiratory activation. This study demonstrates that compounds that enhance the respiratory activity of S. aureus may be potential leads in the development of therapeutic agents for chronic S. aureus-biofilm-related infections. IMPORTANCE Chronic infections caused by Staphylococcus aureus are characterized by biofilm formation, suggesting that methods to control biofilm formation may be of therapeutic value. The small compound JBD1 showed biofilm inhibitory activity and increased sensitivity to aminoglycosides and respiratory activity of S. aureus. Additionally, transcriptomic and metabolomic analyses demonstrated that JBD1 induced metabolic remodeling. All JBD1-induced phenotypes were suppressed by the extracellular addition of an excess amount of menaquinone, indicating that JBD1-mediated respiratory stimulation inhibits biofilm formation and triggers metabolic remodeling in S. aureus. These findings suggest a strategy for developing new therapeutic agents for chronic S. aureus infections.

Dynamic changes in clinical characteristics and serotype distribution of invasive pneumococcal disease among adults in Japan after introduction of the pediatric 13-valent pneumococcal conjugate vaccine in 2013-2019.

Nationwide population-based surveillance for invasive pneumococcal disease (IPD) is being conducted in few Asian countries. We aimed to evaluate the clinical characteristics and serotype distribution among Japanese adult patients with IPD after introduction of the pediatric 13-valent pneumococcal conjugate vaccine (PCV13) in 2013. IPD surveillance was conducted among adults between 2013 and 2019, and 1,995 patients were analyzed by time period (early, 2013-2015; middle, 2016-2017; late, 2018-2019). We found that the period of 2018-2019 was independently associated with a lower risk of fatal outcome, compared with the period of 2013-2015. The proportion of those with serotype PCV13-nonPCV7 decreased significantly in patients aged 15-64 years and in those aged ≥ 65 years within 3 years after the introduction of pediatric PCV13. By contrast, the proportion of those with nonvaccine serotype increased significantly in those aged ≥ 65 years, but not in those aged 15-64 years. No significant change was found in the proportion of 23-valent polysaccharide pneumococcal vaccine (PPSV23)-nonPCV13 in both of adults aged 15-64 years and ≥ 65 years. The proportions of PCV15-, PCV20- and PCV24-covered serotypes were 38%, 56% and 58% in adult patients with IPD aged ≥ 65 years during the late period. Our data on the serotype distribution support an indirect effect from pediatric PCV13 use among adults, and afford a basis for estimates of protection against IPD by vaccination with newly developed PCVs in older adults in Japan.

Staphylococcus aureus utilizes environmental RNA as a building material in specific polysaccharide-dependent biofilms.

Biofilms are surface-bound microbial communities that are typically embedded in a matrix of self-produced extracellular polymeric substances and can cause chronic infections. Extracellular DNA is known to play a crucial role in biofilm development in diverse bacteria; however, the existence and function of RNA are poorly understood. Here, we show that RNA contributes to the structural integrity of biofilms formed by the human pathogen Staphylococcus aureus. RNase A dispersed both fresh and mature biofilms, indicating the importance of RNA at various stages. RNA-sequencing analysis demonstrated that the primary source of RNA in the biofilm matrix was the Brain Heart Infusion medium (>99.32%). RNA purified from the medium promoted biofilm formation. Microscopic and molecular interaction analyses demonstrated that polysaccharides were critical for capturing and stabilizing external RNA in biofilms, which contributes to biofilm organization. These findings provide a basis for exploring the role of externally derived substances in bacterial biofilm organization.

Pneumococcal meningitis in adults in 2014-2018 after introduction of pediatric 13-valent pneumococcal conjugate vaccine in Japan.

We assessed the impact of the pediatric 13-valent pneumococcal conjugate vaccine (PCV13) on pneumococcal meningitis in adults in Japan in 2014-2018 by comparing epidemiological characteristics of adults with invasive pneumococcal disease with (n = 222) and without (n = 1258) meningitis. The annual incidence of pneumococcal meningitis in 2016-2018 was 0.20-0.26 cases/100,000 population. Age (p < 0.001) and case fatality rate (p = 0.003) were significantly lower in patients with meningitis than in those without meningitis. The odds of developing meningitis were higher in asplenic/hyposplenic or splenectomized patients (adjusted odds ratio [aOR] 2.29, 95% CI 1.27-4.14), for serotypes 10A (aOR 3.26, 95% CI 2.10-5.06) or 23A (aOR 3.91, 95% CI 2.47-6.19), but lower for those aged ≥ 65 years (aOR 0.59, 95% CI 0.44-0.81). PCV13 had an indirect effect on nonmeningitis, but its impact on meningitis was limited because of an increase in non-PCV13 serotypes. Of meningitis isolates, 78 (35.1%) and 3 (1.4%) were penicillin G- or ceftriaxone-resistant, respectively. We also confirmed an association of the pbp1bA641C mutation with meningitis (aOR 2.92, 95% CI 1.51-5.65).

Acsbg1-dependent mitochondrial fitness is a metabolic checkpoint for tissue Treg cell homeostasis.

Regulatory T (Treg) cells are critical for immunological tolerance and immune homeostasis. Treg cells strongly rely on mitochondrial metabolism and show a lower level of glycolysis. However, little is known about the role of lipid metabolism in the regulation of Treg cell homeostasis. Some members of the ACSL family of acyl-coenzyme A (CoA) synthases are expressed in T cells, but their function remains unclear. A combination of RNA-sequencing and proteome analyses shows that Acsbg1, a member of ACSL, is selectively expressed in Treg cells. We show that the genetic deletion of Acsbg1 not only causes mitochondrial dysfunction, but it also dampens other metabolic pathways. The extrinsic supplementation of Acsbg1-deficient Treg cells with oleoyl-CoA restores the phenotype of the Treg metabolic signature. Furthermore, this pathway in ST2+ effector Treg cells enhances immunosuppressive capacity in airway inflammation. Thus, Acsbg1 serves as a metabolic checkpoint governing Treg cell homeostasis and the resolution of lung inflammation.

Common Features and Intra-Species Variation of Cutibacterium modestum Strains, and Emended Description of the Species.

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium, which includes Cutibacterium acnes. The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as "Propionibacterium humerusii". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA-DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79-80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense. Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m/z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group "P. humerusii" is included in C. modestum.

α-galactosylceramide-stimulated invariant natural killer T-cells play a protective role in murine vulvovaginal candidiasis by Candida albicans.

BACKGROUND: Vulvovaginal candidiasis is a common superficial candidiasis; however, a host's immunological mechanism against vaginal Candida infection remains unknown. OBJECTIVES: In this study, we aimed to elucidate the effect of iNKT cell activation on vulvovaginal candidiasis. METHODS: Using a vulvovaginal candidiasis model with estrogenized mice, we evaluated the fungal burden and number of leukocyte infiltrations in the vaginal lavage of wild-type C57BL/6J mice after Candida albicans inoculation. One day before C. albicans inoculation, α-galactosylceramide (the α-GalCer group) or sterile phosphate-buffered saline (the sham group) was intraperitoneally injected into the mice. We also evaluated the level of antimicrobial peptide S100A8 in the vaginal lavage and analyzed the correlation between S100A8 concentration and the number of vaginal leukocyte infiltrations. Moreover, the number of uterine and vaginal immune cells were evaluated using flow cytometry. RESULTS: The number of vaginal leukocyte infiltrations was significantly higher in the α-GalCer group than in the sham group 3 days after C. albicans inoculation. In addition, the fungal burden was significantly lower in the α-GalCer group than the sham group at 7 days after inoculation. In the analysis of S100A8 concentration of vaginal lavage, there were no significant differences between these two groups, although S100A8 concentration and the number of vaginal leukocyte infiltrations were positively correlated in the α-GalCer group. Moreover, the number of vaginal iNKT cells, NK cells and CD8+ T-cells was significantly higher in the α-GalCer group 3 days after inoculation. CONCLUSIONS: α-GalCer-stimulated iNKT cells likely play a protective role against vulvovaginal candidiasis.

SCD2-mediated monounsaturated fatty acid metabolism regulates cGAS-STING-dependent type I IFN responses in CD4+ T cells.

Host lipid metabolism and viral responses are intimately connected. However, the process by which the acquired immune systems adapts lipid metabolism to meet demands, and whether or not the metabolic rewiring confers a selective advantage to host immunity, remains unclear. Here we show that viral infection attenuates the expression of genes related to lipid metabolism in murine CD4+ T cells, which in turn increases the expression of antiviral genes. Inhibition of the fatty acid synthesis pathway substantially increases the basal expression of antiviral genes via the spontaneous production of type I interferon (IFN). Using a combination of CRISPR/Cas9-mediated genome editing technology and a global lipidomics analysis, we found that the decrease in monounsaturated fatty acid caused by genetic deletion of Scd2 in mice was crucial for the induction of an antiviral response through activation of the cGAS-STING pathway. These findings demonstrate the important relationship between fatty acid biosynthesis and type I IFN responses that enhances the antiviral response.

Distribution and Variation of Serotypes and Pneumococcal Surface Protein A Clades of Streptococcus pneumoniae Strains Isolated From Adult Patients With Invasive Pneumococcal Disease in Japan.

Pneumococcal surface protein A (PspA) is a surface protein of Streptococcus pneumoniae that may be a candidate antigen for new pneumococcal vaccines. This study investigates the distribution of PspA clades of the causative strains of adult invasive pneumococcal disease (IPD) in Japan. Of the 1,939 strains isolated from cases of adult IPD during 2014-2019, the PspA clades of 1,932 (99.6%) strains were determined, and no pspA was detected in the remaining 7 strains (0.4%). PspA clades 1-6 were detected in 786 (40.5%), 291 (15.0%), 443 (22.8%), 369 (19.0%), 33 (1.7%), and 6 (0.3%) strains, respectively. New PspA clades (0.2%) were identified in two non-typeable and two serotype 35B pneumococci. The proportions of clade 1 and clade 2 showed significantly decreased and increased trends, respectively. Furthermore, the PspA clade of pneumococcal strains was partially serotype- and sequence type-dependent. The majority of strains belonging to serotypes contained in both the 13-valent pneumococcal conjugate vaccine (PCV13) and the 23-valent pneumococcal polysaccharide vaccine (PPSV23) belonged to PspA clades 1 or 3. In contrast, the distribution of clades in non-vaccine serotypes was wider than that of vaccine serotype pneumococci. Our findings demonstrate that almost all pneumococcal strains from adult IPD express PspA clades 1-4, especially for non-vaccine serotypes. These results may be useful for the development of a new pneumococcal vaccine with PspA.

Hierarchical Model for the Role of J-Domain Proteins in Distinct Cellular Functions.

In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and nucleotide exchange factor GrpE. JDPs determine substrate specificity for the Hsp70 system; however, knowledge on their specific role in bacterial cellular functions is limited. In this study, we demonstrated the role of JDPs in bacterial survival during heat stress and the DnaK-regulated formation of curli-extracellular amyloid fibers involved in biofilm formation. Genetic analysis demonstrate that only DnaJ is essential for survival at high temperature. On the other hand, either DnaJ or CbpA, but not DjlA, is sufficient to activate DnaK in curli production. Additionally, several DnaK mutants with reduced activity are able to complement the loss of curli production in E. coli ΔdnaK, whereas they do not recover the growth defect of the mutant strain at high temperature. Biochemical analyses reveal that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the solubilization of MlrA, a DNA-binding transcriptional activator for the csgD promoter. Furthermore, DnaJ and CbpA also keep CsgA in a translocation-competent state by preventing its aggregation in the cytoplasm. Our findings support a hierarchical model wherein the role of JDPs in the Hsp70 system differs according to individual cellular functions.

Intensive diagnostic management of coronavirus disease 2019 (COVID-19) in academic settings in Japan: challenge and future.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first emerged in Wuhan, China, and has spread globally to most countries. In Japan, the first COVID-19 patient was identified on January 15, 2020. By June 30, the total number of patients diagnosed with COVID-19 reached 18,000. The impact of molecular detection of pathogens is significant in acute-care settings where rapid and accurate diagnostic measures are critical for decisions in patient treatment and outcomes of infectious diseases. Polymerase chain reaction (PCR)-based methods, such as quantitative PCR (qPCR), are the most established gene amplification tools and have a comprehensive range of clinical applications, including detecting a variety of pathogens, even novel agents causing emerging infections. Because SARS-CoV-2 contains a single-stranded RNA genome, reverse-transcription qPCR (RT-qPCR) has been broadly employed for rapid and sensitive quantitative measurements of viral RNA copy numbers. The RT-qPCR method, however, still requires time-consuming reactions with two different enzymes in addition to isolation of RNA from patient samples, limiting the numbers of testing institutions for diagnosing SARS-CoV-2 infection. Japan is known to have performed a relatively small number of PCR tests as well as confirmed cases among developed nations; as of June 30, 2020, approximately 390,000 people in Japan had undergone PCR tests. Given the devastating impact on medical services and the scale of demand for diagnostic testing of COVID-19, it has been proposed that academic settings such as basic research departments in university/college can be engaged in diagnosing, especially in university hospitals or academic medical centers. In collaboration with established diagnostic laboratories, academic facilities can divert their function to detecting virus from patients with suspected COVID-19, adopting existing specialized expertise in virus handling, molecular work, and data analysis. This in-house testing strategy facilitates the rapid diagnosing of thousands of samples per day and reduces sample turnaround time from 1 week to less than 24 h. This review provides an overview of the general principles, diagnostic value, and limitations of COVID-19 diagnosis platforms in Japan, in particular in-house testing at academic settings.

Seroepidemiological analysis of anti-pneumococcal surface protein A (PspA) immunoglobulin G by clades in Japanese population.

BACKGROUND: Pneumococcal surface protein A (PspA) is one of the candidates of the novel pneumococcal protein vaccines. The seroepidemiology of naturally acquired anti-PspA immunoglobulin G (IgG) by clades, across a wide range of ages has not been investigated. METHODS: We examined the concentrations of anti-PspA IgG by clades (1, 2, 3, 4, and 5) in 397 sera from persons aged 0-≥70 years by enzyme-linked immunosorbent assay, and determined the geometric mean concentrations (GMCs) by age group. The relationships between concentrations of anti-PspA IgG antibody for each clade for each person were also assessed. RESULTS: GMC of anti-PspA IgG was lowest, highest, and plateaued in those aged 6-11 months, 5-9-years, and 20-49 years, respectively. It gradually declined in those aged > 70 years. GMCs patterns in different age groups were similar for all clades. Correlations were found especially within the same PspA family (between clades 1 and 2 or clades 4 and 5). CONCLUSIONS: Our data suggested that most people acquired anti-PspA IgG across clades 1, 2, 3, 4, and 5 during childhood. These results would be a fundamental data of clade-specific anti-PspA IgG antibodies.

Roles of lytic transglycosylases in biofilm formation and ß-lactam resistance in methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is responsible for numerous community outbreaks and is one of the most frequent causes of nosocomial infections with significant morbidity and mortality. While the function of lytic transglycosylases (LTs) in relation to cell division, biofilm formation, and antibiotic resistance has been determined for several bacteria, their role in S. aureus remains largely unknown. The only known LTs in S. aureus are immunodominant staphylococcal antigen A (IsaA) and Staphylococcus epidermidis D protein (SceD). Our study demonstrates that, in a strain of methicillin-resistant S. aureus (MRSA), IsaA and SceD contribute differently to biofilm formation and ß-lactam resistance. Deletion of isaA, but not sceD, led to decreased biofilm formation. Additionally, in isaA-deleted strains, ß-lactam resistance was significantly decreased compared to that of wild-type strains. Plasmid-based expression of mecA, a major determinant of ß-lactam resistance in MRSA, in an isaA-deleted strain did not restore ß-lactam resistance, demonstrating that the ß-lactam susceptibility phenotype is exhibited by isaA mutant regardless of the production level of PBP2a. Overall, our results suggest that IsaA is a potential therapeutic target for MRSA infections.

Identification of a Novel Variant Form of Aspergillus fumigatus CalC and Generation of Anti-CalC Monoclonal Antibodies.

Aspergillus fumigatus is a critical human fungal pathogen that infects the host via inhalation of airborne conidia. These conidia then germinate to form filamentous hyphae, which secrete various elements to survive in the host lung.Elements such as proteins secreted by A. fumigatus can act as virulence factors in host tissues. Among secreted proteins, we were interested in the thaumatin-like proteins of A. fumigatus. In our analysis of the function of thaumatin-like proteins, we found that, like CalA and CalB, CalC has a secreted form. Originally, CalC was predicted to be a GPI-anchored protein, as documented in the Aspergillus Genome Database. Here, we report on a novel secreted form of CalC. Furthermore, we established two novel hybridomas, C103 and C306, which recognized CalC. Monoclonal antibodies produced by these hybridomas responded to recombinant CalC produced by the mammalian cell line HEK293T and to the supernatant of cultured A. fumigatus.Taken together, our data suggest that calC can be spliced to give rise to a novel secretory form of CalC, which is present in the supernatant of cultured A. fumigatus. The hybridomas that we established will be helpful in understanding the biological role of A. fumigatus CalC.