Microbiology and Immunology最新文献

Enterococci are commensal bacteria in humans that are found in the intestine, pharynx and oral cavity and sometimes cause opportunistic infections such as intra-abdominal infections and bacteraemia. Several enterococcal species, especially E. faecium and E. faecalis, have become clinical problems as drug-resistant bacteria, including vancomycin-resistant Enterococci (VRE). Therefore, new antibacterial agents against enterococcal infections are needed. Because several fatty acids (FAs) have been reported to possess antibacterial activity, in this study, the effects of 3 unsaturated fatty acids on enterococci were evaluated using clinically isolated strains. Among the 3 fatty acids we tested, linoleic acid (LA) and palmitoleic acid (POA) exhibited antibacterial activity against E. faecalis and E. faecium. Next, we evaluated the antibacterial activities of the two FAs against 126 enterococcal clinical strains, including five different species. The minimum inhibitory concentration (MIC) of LA varied among bacterial species and strains (ranging from 37.5 to 1200 μM), while the MIC of POA was similar (ranging from 37.5 to 150 μM). Among species, E. faecium strains presented higher MICs than E. faecalis, E. avium and E. casseliflavus strains. In 6 VRE strains, the MICs of LA showed variable (150-1200 μM), whereas MICs of POA were constant at 75 μM. We subsequently investigated the combined effects of LA and POA with several antibiotics and found that LA had synergistic effects with β-lactam, gentamicin, ciprofloxacin and vancomycin against E. faecalis and E. faecium strains, including VRE, whereas POA did not have a significant effect. Our results indicate the potential application of FAs for enterococcal infection.

Cover photograph: Gene organization of the assembly and insertion machinery (Aim) operon in Porphyromonas gingivalis. Schematic representation of the chromosomal arrangement of genes PGN_0296 to PGN_0301. Arrows indicate the direction of transcription. T9SS: type IX secretion system. Microbiol Immunol: 70:9-14. Article link here

Chromoblastomycosis (CBM) is a chronic skin and subcutaneous infection mainly caused by Fonsecaea pedrosoi, a dematiaceous fungus with various morphotypes. Characteristic sclerotic cells-globe-shaped, multiseptated and pigmented-are found in lesions of infected individuals, though their differentiation in the host remains poorly understood. To evaluate in vitro activity of five antifungal drugs-itraconazole (ITZ), posaconazole (PCZ), voriconazole (VCZ), fluconazole (FCZ), and caspofungin (CAS)-against Fonsecaea spp. conidia or sclerotic cells, assessing their minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) and correlated the ITZ MIC with patients' clinical evolution. Forty-three clinical isolates of Fonsecaea spp. and the F. pedrosoi strain ATCC 46428 were assessed for susceptibility to ITZ, PCZ, VCZ, FCZ, and CAS following Clinical Laboratory Standard Institute guidelines (CLSI) (document M38-A2). MIC values were determined after 5 days of incubation at 30°C, followed by MFC determination, with geometric mean MIC (GMMIC) and MFC (GMMFC) used for comparison. PCZ was the most effective antifungal drug, with geometric mean MICs of 0.3 µg/mL (conidia) and 1 µg/mL (sclerotic), and MFCs of 2.98 and 6.72 µg/mL, respectively. Clinical follow-up revealed that higher ITZ MIC values (0.9 µg/mL) correlated with poor patient outcomes compared to lower values in improved or cured patients. These findings highlight PCZ and VCZ as promising options for CBM treatment, especially for patients not responding to ITZ.

Orthohantaviruses are a genus of zoonotic viruses from the family Hantaviridae, which is primarily known for their ability to cause severe human diseases. The virus is primarily known for causing two major human diseases, hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. However, the frequency of this infection remains largely unknown. Hence, the aim of this study was to diagnose patients infected with Orthohantavirus in India. We tested serum samples from 216 febrile patients, presented at Lisie Hospital, Kochi, for positive anti-orthohantavirus IgM and IgG antibodies. All the participating patients were negative for dengue and scrub typhus IgM antibodies. In total, 16.20% (n = 35) of the febrile patients were positive for anti-orthohantavirus IgM and 11.57% (n = 25) for anti-orthohantavirus IgG antibodies. Anti-orthohantavirus IgM and IgG both were detected in 4.63% (n = 10) of the enrolled subjects. In total, 51.43% of the IgM-positive patients had a final diagnosis of either viral fever or acute febrile disease, with only two patients reported to have contact with rodents, and none of these patients had traveled outside of their place of residence. Orthohantavirus infections may be endemic in India, as their hosts are omnipresent. However, the lack of proper diagnostic tools and limited awareness of these emerging infections amongst doctors in India reduces the diagnosis of the disease. Therefore, there is a pressing need for educating healthcare providers about the circulation of orthohantaviruses in India. There is an urgent need for the development of serotype-specific, affordable, and accurate diagnostic tools for early diagnosis of orthohantavirus infections.

HEV causes chronic infections that are detrimental to immunocompromised patients. Previous studies showed alterations of γδ T cell subsets at the acute phase of HEV infection. To assess a possible role of CMV, we have examined the frequencies and responses to CMV and HEV of blood γδ T cell subsets from control donors and acute-phase HEV patients with or without CMV. CMV DNA was mostly undetectable in the blood of CMV-seropositive HEV patients, and anti-CMV antibodies were only slightly elevated. However, Vγ9^negVδ1^pos cells were enriched in vivo, suggesting an increased CMV burden. In contrast, γ9δ1 cells were depleted in most HEV patients, regardless of CMV status. Culturing with IL-2 and IL-15 led to strong γ9δ1 T cell enrichment in samples from HEV patients. After IL-2/IL-15 sensitization, analysis of IFN-γ responses to CMV-infected fibroblasts or hepatocarcinoma cells (with IL-18) showed innate responsiveness to CMV in γ9δ1, γ9δ2, and γ9^negδ1^pos cells in some control subjects and CMV-seronegative patients. However, these responses were selectively exacerbated in CMV-seropositive HEV patients, who also showed significant αβ T cell responses to CMV. This indicates reactivation of anti-CMV immunity. Responses to HEV-infected HepG2 cells remained undetected. IFN-γ responses were not associated with TCR downmodulation in γ9δ2 or γ9^negδ1^pos cells. However, IFN-γ-producing γ9δ1 from HEV patients were characterized by high TCR expression, which was downmodulated after stimulation with CMV-infected fibroblasts or CMV/HEV-coinfected HepG2 cells. We conclude that γ9δ1 T cells are selectively mobilized in HEV patients and can be activated by CMV.

Psoriasis is a common immune-mediated skin disorder. miR-34a, as a significant regulatory component, has been involved in the regulation of immune-inflammatory responses. Previous studies have shown that miR-34a is abnormally expressed in psoriasis cell models. However, the role of miR-34a in psoriasis remains unvalidated by any existing studies. We co-stimulated HaCaT cells with IL-17A, IL-22, TNF-α, IL-1α, and M5 to establish an in vitro model of psoriasis. qPCR was used to detect the expression of miR-34a; ELISA was performed to measure the secretion levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17, and IL-23 in HaCaT cells; CCK-8 and EdU staining assays were used to assess cell growth and proliferation; Western blot analysis was employed to assess the expression levels of relevant proteins, including SIRT1, NLRP3, ASC, pro-Caspase-1, and Caspase-1; dual-luciferase reporter gene assays and RNA immunoprecipitation (RIP) were conducted to investigate the interaction between miR-34a and SIRT1. Additionally, a mouse psoriasis model was induced by imiquimod (IMQ) to validate the role of miR-34a in vivo. qPCR results found that miR-34a was upregulated in HaCaT cells. ELISA results indicated that the levels of IL-1β, IL-6, IL-17, and IL-23 were significantly elevated in the M5-treated group. CCK-8 and EdU staining assays revealed that M5 treatment notably increased the proliferation and viability of HaCaT cells. After treatment with the miR-34a inhibitor, the levels of cytokines and cell proliferation were significantly reduced. Western blot analysis showed that M5 treatment resulted in a significant decrease in SIRT1 protein levels, while the expression levels of NLRP3, ASC, pro-Caspase-1, and Caspase-1 were increased. In contrast, miR-34a-inhibitor treatment showed the opposite results. Dual-luciferase reporter and RIP assays indicated a negative feedback interaction between miR-34a and SIRT1. Rescue experiments showed that overexpression of SIRT1 effectively inhibited NLRP3 expression, significantly reduced IL-23/IL-17 immune-inflammatory responses, and suppressed HaCaT cell proliferation, while overexpression of NLRP3 had the opposite effects. In the IMQ-induced mouse psoriasis model, the IMQ group showed severe psoriasis-like phenotypes, obvious skin pathological changes, upregulated miR-34a expression, and increased skin cytokine levels; all these abnormalities were alleviated by miR-34a inhibitor treatment. miR-34a mediates the IL-23/IL-17 immune-inflammatory response and promotes HaCaT cell proliferation by regulating the SIRT1/NLRP3 pathway.

Cattle exhibit distinct immune system features, including ultra-long IGH CDR3 regions, which are characteristic of their adaptive immunity. However, less is known about the diversity and functional aspects of their T cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) repertoire. This study aimed to investigate the features of the TCR β chain CDR3 repertoire in cattle using high throughput sequencing. The spleen tissue samples were collected from cattle, and the TCR β CDR3 repertoires were constructed using high-throughput sequencing. Key attributes analyzed included CDR3 length distribution, V and J gene usage, sequence diversity, and specific amino acid enrichment. The analysis also incorporated the impact of recombination signal sequences (RSS) quality and the physical distance on the rearrangement of germline genes. Frequently utilized V and J genes included TRBV7, TRBV6, TRBV14, TRBV21, TRBV29, TRBV5, TRBJ2-2, and TRBJ1-2. A bias in TRBV gene rearrangement was identified, influenced by RSS quality, where genes with RSS information content (RIC) scores below -45 showed reduced rearrangement frequency. The CDR3 length distribution displayed a bell-shaped curve, with a preference for the amino acids serine (S), glycine (G), and alanine (A). Compared to humans and mice, cattle exhibited lower diversity in their TCR β CDR3 repertoire and a higher proportion of dominant clones. This study provides insights into the TCR β CDR3 repertoire in cattle, enhancing our understanding of the species' adaptive immune system. These findings offer valuable insights for future research on the immune repertoire in cattle.

Orthobornaviruses express X and the phosphoprotein (P) from a bicistronic X/P mRNA, and these proteins regulate polymerase activity. In mammalian orthobornaviruses, the 5' untranslated region (5' UTR) of the X/P mRNA controls the translational balance between X and P and thereby promotes efficient replication. Avian bornaviruses (ABVs) belong to two clades, clade-2 and -3, that differ in the structure and length of the 5' UTR of the X/P mRNA. However, the functional consequences of these differences remain unclear. Using reverse genetics, we generated chimeric viruses by reciprocally exchanging the 5' UTR of the X/P mRNA among clade 2 parrot bornavirus 5 (PaBV-5) and aquatic bird bornavirus 1 (ABBV-1) and clade 3 PaBV-4. In PaBV-5, a long 5' UTR with a stem-loop and an upstream ORF was required to maintain the X-to-P translational balance. On the other hand, replacing the 5' UTR of the X/P mRNA from PaBV-5 with that from PaBV-4 reduced X expression and markedly impaired viral growth. However, PaBV-4 tolerated the 5' UTR of the X/P mRNA from PaBV-5 without detectable effects on translation or replication, which suggests that translation of PaBV-4 X/P mRNA does not depend on the origin of the 5' UTR. Furthermore, ABBV-1 replicated efficiently with the 5' UTR of the X/P mRNA from PaBV-5 but was strongly attenuated with that from PaBV-4. Taken together, these results demonstrate a clade-dependent requirement for the 5' UTR for translation of the X/P mRNA and provide novel insights into the evolution of translational control in orthobornaviruses.

Bordetella has a type III secretion system that secretes virulence proteins crucial to the establishment of infection. The genes encoding components of the Bordetella type III secretion system are located in the bsc region on the chromosome. This region includes the bcrH1 and bcrH2 genes, which respectively encode the proteins BcrH1 and BcrH2. In this study, we analyzed the functions of BcrH1 and BcrH2 in the Bordetella type III secretion system. First, we created a BcrH1-deficient strain and a BcrH2-deficient strain. We analyzed the amounts of the type III secreted proteins BopB and BopD, which make a complex that forms pores in the host membrane, in bacterial cells of each protein-deficient strain. The results showed that the BopB and BopD signals were weakened in the whole cell fraction of the BcrH1-deficient strain and the BcrH2-deficient strain, respectively. The hemolytic activity and cell toxicity of each BcrH protein-deficient strain were significantly lower than those of the wild-type strain. When anti-BcrH1 and anti-BcrH2 antibodies were used for the immunoprecipitation assay, the BopB and BopD signals were detected in the precipitated fractions, respectively. These results strongly suggest that BcrH1 and BcrH2 are specific chaperones for maintaining the stability of BopB and BopD, respectively.

Oz virus (OZV) is a tick-borne, six-segmented, negative-strand RNA virus in the genus Thogotovirus, family Orthomyxoviridae. A fatal human infection was reported in Japan in 2023. In this study, we established a reverse genetics system to generate infectious recombinant OZV. Six plasmids encoding the full-length OZV genome segments under the control of a murine RNA polymerase I promoter, together with four plasmids expressing viral proteins essential for polymerase activity, were cotransfected into murine cells. This approach enabled efficient recovery of infectious OZV. The recovered recombinant virus exhibited replication kinetics comparable to wild-type OZV. This system provides a platform for molecular studies of OZV.