Medical Microbiology and Immunology最新文献

Clostridium innocuum, a member of the human gut microbiome with intrinsic resistance to vancomycin, has been increasingly associated with inflammatory bowel diseases (IBD). Clinical observations indicate that co-infection with Clostridioides difficile and C. innocuum could lead to poorer clinical remission in ulcerative colitis; however, the pathogenic mechanism of C. innocuum remains unclear. Here, we investigated the effects of vancomycin and C. difficile on C. innocuum secretomes and the functions of the modified secretomes on C. innocuum pathogenicity. The results indicated that, compared to co-culturing with C. difficile, vancomycin was more effective in stimulating the secretion of proteins without a signal peptide, whereas C. difficile was better at promoting the secretion of classical secretory proteins. Based on these results, we further analyzed the effects of three abundant classical secretory proteins on C. innocuum virulence utilizing recombinant proteins. The results demonstrated that the NlpC/P60-containing protein (NlpC/P60) can enhance C. innocuum biofilm formation and adherence to HT-29 cells. Additionally, NlpC/P60, D-Ala-D-Ala carboxypeptidase, and a polysaccharide deacetylase were able to stimulate IL-8 production of HT-29 cells and TNF-α production of Raw264.7 macrophages. Additionally, recombinant NlpC/P60 and polysaccharide deacetylase exhibited cytotoxicity on Raw264.7 cells at 48 h. As the production of IL-8 and TNF-α is closely associated with IBD development, it is suggested that C. innocuum secretomes, under the influence of vancomycin or C. difficile, could contribute to IBD progression by enhancing inflammation and host-pathogen interactions.

Community-acquired pneumonia (CAP) is a significant health threat for adults. Although conjugate vaccines have reduced pneumococcal CAP incidence in children, Streptococcus pneumoniae-related CAP remains prevalent among older adults. The nasopharynx acts as a reservoir for S. pneumoniae, yet the interplay between this pathogen and the nasopharyngeal microbiome during and after pneumonia remains poorly understood. This study included 61 adult patients diagnosed with pneumococcal CAP and 61 matched healthy controls. An S. pneumoniae-specific PCR, urine antigen tests and bacterial cultures were performed. Nasopharyngeal swabs collected at admission and three months post-infection were analyzed for microbiome dynamics through 16 S rRNA gene amplicon sequencing. 16 S rRNA gene amplicon sequencing revealed Streptococcus spp. in the majority of all nasopharyngeal samples during infection compared to the other diagnostic test performed. While overall bacterial biomass did not differ between groups, patients exhibited higher alpha diversity (p = 0.012) and lower microbiome stability post-infection. Beta diversity analysis distinguished infection from healthy status (p = 0.002). Taxonomic analysis showed similar core microbiota across groups, but Streptococcus spp. was significantly more abundant during infection, particularly in those patients with viral co-infections. Notably, unique significant bacterial interactions were identified both during and after infection, as well as in healthy states. A negative correlation was observed between Corynebacterium and Streptococcus spp. in infected patients, suggesting a potential antagonistic interaction between these taxa. The nasopharyngeal microbiome in patients with pneumococcal CAP demonstrates persistent disruption post-infection, characterized by lower resilience three months after acute illness. Additionally, we identified specific bacterial interplays during and after infection that differed from those in healthy donors. These bacterial dynamics might play critical roles in pathogen colonization resistance and infection prevention. Thus, our findings highlight the need for further investigation into microbial interactions and potential microbiome-based therapies for respiratory infections, particularly in vulnerable populations.

Leishmania donovani is the causative agent of the fatal visceral leishmaniasis (VL) disease in humans in the tropical regions, mainly the Indian Subcontinent and Africa. We have previously described centrin1, a basal body associated cell division specific protein in this parasite important for the parasite's host intracellular stage. In this study, we identified a novel centrin1-binding protein called LdDRP through pull-down and MS/MS analysis, which is a homolog of the XPC protein of humans involved in DNA damage. The protein interaction with LdCen1 was also confirmed through peptide spectrum analysis against the UniProt database. Immunofluorescence analysis confirms that LdDRP is localized within the nucleus, suggesting the protein's possible role in DNA interaction. The overexpression of three LdDRP forms in the parasite, each fused with HA-tag (LdDRPF [full length] LdDRPN [only N-terminal], and LdDRPC [only C-terminal]), revealed that only LdDRPF and LdDRPC were able to support the retention of the parasite's shape and promote rapid division following the UV-damage recovery period. This was also correlated to the elevated expression level of both LdDRPC and LdCen1, by Western blot analysis soon after UV-C exposure in the parasites compared to control. The study emphasizes the role of the LdDRP, and its crucial domains involved in the DNA binding process, DNA damage response, and interaction with centrin, particularly in response to UV-C light-induced DNA damage.

Group A Streptococcus (GAS) is a pathogen that is capable of colonizing various infection sites and can potentially elicit an inadequate immune response that will lead to sepsis. The processes underlying this misdirected immune reaction remain poorly understood, and reliable biomarkers for indicating impending organ failure during sepsis are still missing. The present study aims to identify parameters that can predict the onset of end-organ damage in the course of sepsis. To that extent, we investigated key aspects of the immune response in early-phase sepsis following infection of different tissues in a mouse model, using Brefeldin A to link cytokine production to specific cell types through multi-parameter flow cytometry. Subcutaneous and intravenous GAS infections resulted in clinical sepsis, which was paralleled by peripheral blood lymphopenia. Intravenous infection in particular was associated with a higher bacterial burden in the liver that strongly correlated with an increased granulocyte-to-lymphocyte ratio of the peripheral blood. Strikingly, IL-6 overexpression was more pronounced in intravenous infection and strongly correlated with hepatic stress, indicated by elevated bacterial loads in the liver. Collectively, our data highlight the potential utility of IL-6 in conjunction with an elevated granulocyte-to-lymphocyte ratio as promising early indicators of concomitant liver stress in sepsis.

We employed a CRISPR/Cas9 technique in Leishmania major to evaluate its efficiency in editing a kDNA-associated gene, the universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division. Using this toolkit, we generated UMSBP mNG-tagged and single knockout L. major (LmUMSBP^+/-) parasites, which were confirmed by PCR, confocal microscopy and Western blot analyses. The growth rate of promastigotes in culture and their infectivity in macrophages were analysed in vitro. Mice were immunized with the LmUMSBP^+/- mutant strain, and lesion size and parasite burden were measured upon challenge with wild-type (WT) L. major. Cytokines were quantified in supernatants of lymph node cell cultures. The results suggested successful expression and localization of the UMSBP mNG-tagged protein within the kinetoplast in both promastigote and intracellular amastigote forms, confirming the consistency of fluorescence tagging throughout various stages of the Leishmania life cycle. Attenuated LmUMSBP^+/- parasites showed significantly reduced growth in culture (P < 0.05), increased apoptosis (P < 0.05) and downregulation of tryparedoxin peroxidase (TXNPx) and trypanothione synthetase (TryS) gene expression compared to WT L. major. LmUMSBP^+/- mutant strains did not cause lesions in a susceptible BALB/c mouse model. Furthermore, immunization with LmUMSBP^+/- parasites elicited a Th1 immune response, characterized by significantly higher IFN-γ and lower IL-4 production in cell culture (P < 0.001), which was associated with partial protection against WT L. major challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. In this study, we successfully validated a practical CRISPR/Cas9 toolkit in L. major, targeting the kinetoplast-associated gene UMSBP. Our findings suggest that the UMSBP single-allele knockout mutant holds promise as a valuable tool for studying the role of the kinetoplast in Leishmania biology and as a potential candidate for further investigation as a live-attenuated vaccine against Leishmania infection.

Burkholderia cenocepacia, an opportunistic pathogen, poses a significant threat to human health, necessitating the discovery of effective quorum sensing inhibitors (QSIs). In this study, the quorum sensing inhibitory effects of deferiprone (DFP) on the B. cenocepacia 162,638 were validated. Notably, DFP demonstrated an ability to inhibit and disrupt bacterial biofilms, reducing biofilm formation by 44.59% at 1/4 MIC (minimum inhibitory concentration) and 24.32% at 1/8 MIC concentrations. The study also investigated DFP's impact on motility, virulence, and QS signal levels. LC-MS/MS analysis showed a gradual reduction in the QS molecule C6-HSL as DFP concentrations increased. Additionally, DFP's non-hemolytic properties and safety profile, as verified in Galleria mellonella infection models, highlighted its biocompatibility. RT-qPCR results further indicated that DFP downregulated QS-related gene expression, particularly those involved in ferric uptake regulation protein (Fur). Molecular docking studies identified Fur as a key target for DFP's inhibitory action. Collectively, DFP was shown as a potential QSI with practical applications for controlling B. cenocepacia infections.

Whether the magnitude of individual cytokine, cellular, and clinical responses to the intravenous lipopolysaccharide (LPS) challenge is constant in individuals over extended time periods is unknown. Nine healthy volunteers received an intravenous LPS injection of 2 ng/kg bodyweight twice at intervals of at least one year. Circulating cytokines and leukocyte subsets were quantified using a multiplex immunoassay and cytometry by time-of-flight, respectively. Self-reported symptoms and vital signs were also assessed. We observed moderate to strong intra-individual correlations in the responsiveness of most cytokines (IL-6 [AUC_0 - 10]: R = 0.93, p < 0.001; CRP [mg/dL]: R = 0.88, p = 0.004; IL-8 [AUC_0 - 10]: R = 0.71, p = 0.031; TNF-alpha [AUC_0 - 10]: R = 0.67, p = 0.047; IL-10 [AUC_0 - 10]: R = 0.42, p = 0.26) and cellular subsets (CD8 T lymphocytes: R = 0.9, p = 0.002; B lymphocytes [G/L]: R = 0.89, p = 0.003; CD4 T lymphocytes: R = 0.84, p = 0.001; neutrophils: R = 0.80, p = 0.017; monocytes: R = 0.16, p = 0.710) between the 1st and 2nd LPS challenges. Vital signs and symptoms were not reproducible. While the average cellular and clinical response was similar between the two LPS challenges, we found a significantly attenuated AUC_0 - 10 of IL-6 (percent difference, -41.9% [95% CI -73.0 - -10.7]) and TNF-alpha (percent difference, -35.7% [95% CI -70.0 - -1.6]) at the 2nd LPS challenge. Individual cytokine and cellular responses to intravenous LPS showed a significant degree of correlation when measured more than one year apart. These correlations did not translate to the reproducibility of clinical symptoms and vital signs, which showed greater variability and were not constant over time. The partly reduced cytokine release in the 2nd LPS challenge might be interpreted as an indicator of a long-lasting tolerance to endotoxin.

Dengue virus (DENV) is a global health threat, with approximately 390 million infections annually, ranging from mild dengue fever to severe dengue hemorrhagic fever and shock syndrome. MicroRNA (miRNA) are crucial post-transcriptional regulators which may regulate host resistance to DENV infection. This study aimed to identify miRNAs involved in natural resistance to DENV infection. Individuals from a dengue-endemic area were classified as susceptible (SD) or resistant (RD) according to their anti-DENV antibody status. RD individuals were seronegative despite high local DENV infection prevalence. Monocytes susceptibility to DENV infection was assessed in vitro. The miRNome profiles of the monocytes from 7 individuals per group were assessed upon mock or DENV-2 infection. The antiviral effect of differentially expressed miRNAs was analyzed using miRNA mimics in HeLa cells followed by infection with DENV-1, DENV-2, DENV-3, and DENV-4 serotypes. We performed RNA-seq on miRNA mimic-transfected cells to identify miRNA-targeted genes interacting with DENV proteins. Monocytes from RD individuals exhibit lower DENV-2 production in vitro. The miRNAs miR-155, miR-132-3p, miR-576-5p were overexpressed in monocytes from RD group upon DENV-2 infection. The transfection of miR-155-5p mimic reduced DENV infection and viral production in HeLa cells, regulating 18 genes interacting with DENV proteins and downregulating target genes involved in interferon response, TP53 regulation, apoptosis, and vesicle trafficking (e.g. HSD17B12, ANXA2). Therefore, we show that monocytes from RD individuals show a distinct miRNA expression profile and reduced viral production. In vitro miR-155-5p upregulation induces an antiviral state, revealing potential therapeutic targets to treat dengue.

Leptospirosis is a major cause of acute febrile illness, often presenting with non-specific symptoms that can lead to misdiagnosis. Early laboratory diagnosis is essential for confirmation to avoid misdiagnosis and ensure appropriate management. This study aimed to identify and produce a recombinant protein, approximately 25 kDa, with high antigenicity for diagnostic applications. The 25 kDa protein from Leptospira interrogans was identified as electron transfer flavoprotein beta subunits (Etfβ) and exhibited 98% nucleotide and 99% amino acid homology to the reference strain. Lateral flow assays (LFAs) using recombinant Etfβ (rEtfβ) as antigens were developed to detect specific antibodies, namely rEtfβ-IgM and rEtfβ-IgG, and evaluated their performance against the standard microscopic agglutination test (MAT). Testing 33 paired serum samples from confirmed leptospirosis cases and 24 controls revealed sensitivities of 69.7% for IgM and 57.6% for IgG. However, the combined assays yielded enhanced diagnostic accuracy, achieving a sensitivity of 94.0%, specificity of 95.8%, positive predictive value of 96.9%, negative predictive value of 92.0%, and percent agreement of 94.7% (kappa value of 0.89). Also, the combined LFAs demonstrated 66.7% in initial serum samples whose MAT results were negative, enhancing the capacity for early diagnosis. In conclusion, the developed rapid tests demonstrated strong diagnostic capability, particularly in early-phase leptospirosis, distinguishing between initial and recurrent infections. Importantly, rEtfβ-IgG identified a subset of patients lacking detectable IgM. Thus, integrating rEtfβ-IgM and rEtfβ-IgG is recommended to improve sensitivity and accuracy in endemic populations. The rEtfβ is a promising target for future antigen-based diagnostic strategies for leptospirosis. The rEtfβ antigen shows promise as a target for future development of antigen-based diagnostic strategies for leptospirosis.