Medical Microbiology and Immunology最新文献

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.

It is now widely accepted that lungs are colonized by diverse microbes. Dysbiosis of the lung microbiota has been found to affect the progression of lung cancer. Fungi are a major component of the lung microbiota. However, the causal links between the mycobiome or specific species and lung cancer remain unclear. To address this, we conducted a study examining the composition of lung mycobiota in Non-Small-Cell Lung Cancer (NSCLC) patients using shotgun metagenomics. The differential taxa between NSCLC patients and non-cancer controls were defined by the Wilcoxon rank-sum test. Nested PCR was used to measure the abundance of specific fungal species. Metabolomics analysis was performed to investigate the metabolic reprogramming of macrophages triggered by intracellular infection of specific fungal species. In vitro and in vivo assays were conducted to examine the effect of the specific fungus on cancer cell growth. Our findings showed that Ascomycota, Microsporidia and Mucoromycota were the dominant fungal taxa in the lungs. Talaromyces marneffei (T.marneffei) was the most significantly differential fungus between lung cancer patients and non-cancer controls, with its abundance positively correlated with lung cancer. The lung cancer animal model demonstrated that T.marneffei promotes lung cancer growth. Our study also demonstrated that T.marneffei promotes lung cancer cell growth by inducing dose-dependent M2 macrophage polarization through arginine-ornithine-cycle activation. Furthermore, inhibition of arginase can reduce M2 polarization of macrophages and the survival of T. marneffei inside macrophages. In summary, our study reveals that the increased abundance of T. marneffei in the lungs affects lung cancer cell growth by triggering arginine-induced M2 polarization of macrophages. These findings provide potential drug targets for the development of therapies aimed at targeting the survival of fungi inside macrophages in the fight against cancer.

Heat-labile toxin (LT) from enterotoxigenic Escherichia coli (ETEC) is an important pathogenic protein. Anti-LT antibodies (Abs) induced by vaccination can neutralize the toxin and potentially prevent diarrheal secretion from ~ 60% of ETEC strains expressing LT. However, only superficial investigation of the anti-toxin response is usually conducted in clinical trials. Here, we utilized human serum samples from two clinical trials performed to assess safety, immunogenicity and protection in a controlled human infection model with a LT + ST + CFA/I + H10407 ETEC strain. These Phase 1 and Phase 2b clinical trials explored a prototype ETEC adhesin (CfaE) and a chimeric adhesin-toxoid protein (dscCfaE-CTA2/LTB5) delivered intradermally or transcutaneously with a mutated form of LT (mLT) as an adjuvant. Serum samples were tested for antigen-specific IgG or IgA Abs by immunoblot, enzyme-linked immunosorbent assay (ELISA), or functional neutralizing Abs using LT holotoxin, LTA or LTB subunits. Abs to both LT subunits were present, but the response to each was altered by vaccine formulation, dose, and delivery routes as well as subject. The anti-LT IgG response correlated best to neutralizing antibodies and protection from H10407 controlled challenge when compared to other measures including serum IgA or anti-fimbriae (CfaE) Abs. In addition, our results helped to explain cohort attack rate differences in naïve unvaccinated participants and we found higher anti-LTA IgG post-challenge significantly related to ETEC severity score. Thus, strategies generating and measuring immunity to the complete AB₅ structure of LT and subunits are better determinant of assessing protective immunity against LT + or LT + ST + ETEC diarrheal secretion in humans.

In this paper we report a human ex vivo model of Aspergillus fumigatus and Candida albicans infection of the cornea. We demonstrate the ability to monitor fungal growth on the cornea through optical and immunofluorescence microscopy. Additionally, we establish a method for collection of fungal antigens in polyethersulfone (PES) membrane-based sample collectors from the surface of fungal inoculated corneas. Immunofluorescence microscopy was performed directly on both corneal tissue and PES membrane sample collectors. For the latter case, we devised an original ad-hoc method by attaching the membranes used for the collection on standard glass slides, which we call glass slide KIT. This enabled easy handling and improved efficiency for specifically recognizing the corresponding fungal infections by simple immunoassay protocols. As a result, we firstly observe the ability to monitor fungi in corneal tissue, and secondly demonstrate the adsorption of fungal antigens onto PES membranes. Thus, we report for the first time the specific detection of fungal corneal infections in easy to use PES membrane based glass slide KITs through simple immunofluorescence, as an alternative to microbiological culture.

Aspiration pneumonia is a serious respiratory condition, which is particularly prevalent in patients with dysphagia, neurological disorders, or those undergoing surgical interventions. The formation of multispecies biofilms in the oral cavity, involving the bacterial periodontopathogen Porphyromonas gingivalis and the opportunistic pathogenic fungus Candida albicans, may also be related to the development of this serious disease, contributing also to the resistance to standard antimicrobial treatment. Therefore, this research aimed to evaluate the efficacy of selected antibiotics‒levofloxacin, metronidazole, meropenem, vancomycin‒and antifungal agents‒amphotericin B, caspofungin, and fluconazole‒on these mixed biofilms in the aspiration pneumonia model. While metronidazole and levofloxacin effectively inhibited bacterial viability in the mixed biofilms, lower doses increased release of bacterial proteases. In the conditions of mixed biofilms meropenem and vancomycin showed reduced efficacy, requiring significantly higher doses to achieve similar effect in mixed biofilms as in single bacterial cultures. Treatment with antifungals revealed that amphotericin B significantly impacted fungal cell viability within mixed biofilms, and this effect was enhanced when the antifungal drug was applied in the presence of P. gingivalis. Caspofungin and fluconazole showed variable efficacy, with caspofungin being more effective against C. albicans cells within biofilm.These findings indicated that due to the mutual microbial protection in the mixed-species biofilm, P. gingivalis retained its virulence despite increasing antibiotic doses. However, no excessive benefit of mixed biofilms was observed for C. albicans in the presence of antifungals, indicating the minor importance of yeasts in aspiration pneumonia development and their protective role for other pathogens in mixed-species infection.