Infection and Immunity最新文献

Considerable effort has focused on identifying alternatives to mouse models in research studies. In the field of bacterial pathogenesis, Galleria mellonella and epithelial cell lines have been widely used for this purpose, but the concordance of these models with mice remains unclear. To begin to address this knowledge gap, we used 105 clinical isolates of Pseudomonas aeruginosa for which virulence had previously been determined in a mouse bacteremia model. A semistrong correlation was observed between G. mellonella median time to 50% mortality and mouse 50% pre-lethal dose (LD₅₀) values (Spearman's rank correlation coefficient [ρ] = 0.75), whereas percent A549 epithelial-like cell lysis during co-culture showed a weak correlation to mouse LD₅₀ values (ρ=-0.47). Given the stronger correlation between G. mellonella and mouse virulence, we next examined whether G. mellonella could substitute for mice when asking questions about the virulence of large numbers of P. aeruginosa isolates. Results from mice indicated that isolates with resistance to more antibiotics were significantly less virulent, and the use of G. mellonella identified the same inverse correlation. Furthermore, both models found no evidence for the existence of hypervirulent clonal lineages. In particular, isolates belonging to sequence types defined as high-risk clones were not consistently more virulent than other isolates, despite the known association of high-risk clones with poor clinical outcomes. These findings suggest that G. mellonella can serve as an adequate substitute for mice when addressing specific population-based virulence questions, although conclusions should be confirmed in mice.

Macrolide antibiotics are commonly prescribed to treat Haemophilus influenzae respiratory tract infections. Studies have primarily focused on emerging H. influenzae strains with acquired macrolide resistance, while the bacterium's intrinsic resistance to antibiotics has been underexamined. Here, we used a genome-wide approach of transposon insertion-site sequencing to screen an H. influenzae mutant library grown in sub-inhibitory doses of the macrolide antibiotic clarithromycin (CLR) to identify 33 genes involved in intrinsic CLR resistance. Almost half of these genes are also needed for survival in the mouse lung. We focused on candidate genes necessary for both intrinsic macrolide resistance and lung survival. Two of these genes affect the outer-membrane composition of H. influenzae, orfH and omp26. Deletions of these genes in Rd and nontypeable H. influenzae clinical isolates, Hi375 and NT127, conferred sensitivity to CLR and polymyxin B and increased membrane permeability to ethidium bromide (EtBr). The omp26 mutant was sensitive to killing by human serum. Deletions of orfH or omp26 in an acrR mutant strain overexpressing a multidrug efflux pump abrogated resistance of the acrR mutant to CLR and restored permeability to EtBr. Thus, deletion of these genes not only mitigates the effects of an acquired resistance mechanism but also remarkably overrides it. Complementation of these deletion mutations restored CLR resistance and decreased permeability to EtBr. Our results indicate that the subset of genes with dual roles in intrinsic resistance and host lung survival may provide potential novel combination antimicrobial therapeutic targets.

The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals with compromised immune systems. The ability of the fungus to cause disease depends on key cell-surface features, such as a polysaccharide capsule that protects it from the mammalian immune system. However, the mechanisms by which C. neoformans traffics polysaccharide capsule, melanin, and other materials to the cell surface are poorly understood. In this study, we employed mutants lacking specific subunits of the adaptor protein complex 1 (AP-1) to investigate its role in the elaboration of virulence-related materials at the cell surface. Importantly, the mutants displayed multiple defects, including alterations in capsule size and cell morphology, and defects in melanin production and urease secretion. Together, these results support the key observation that the AP-1 complex is required for C. neoformans survival in phagocytic cells. Together, our findings provide insights into the endomembrane trafficking machinery required for fungal pathogenesis.

Pseudomonas aeruginosa is a common opportunistic pathogen that causes chronic lung infections in individuals with cystic fibrosis. Despite advances in therapies that restore cystic fibrosis transmembrane conductance regulator function, persistent colonization of the airway remains a major clinical challenge. Reduced clearance of P. aeruginosa from the cystic fibrosis airway has been associated with the increased activity of histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase that decreases microtubule acetylation and stability. In this study, we investigated the role of HDAC6 in modulating interactions between P. aeruginosa and cystic fibrosis airway epithelial cells. Pharmacologic inhibition of HDAC6 significantly reduced bacterial adherence in both mouse and human cystic fibrosis epithelial cells. Genetic deletion of HDAC6 produced similar effects, while knockout of a microtubule-stabilizing protein increased bacterial adherence, mimicking the cystic fibrosis phenotype. HDAC6 inhibition also reduced bacterial internalization, although to a lesser extent compared to adherence. These results suggest that microtubule destabilization contributes to the enhanced colonization of cystic fibrosis airways by P. aeruginosa. Targeting host microtubule regulatory pathways, particularly by inhibiting HDAC6, may represent a promising host-directed strategy to limit early bacterial attachment and reduce the risk of chronic infection in cystic fibrosis.

Staphylococcus aureus is a leading cause of bacterial infections worldwide and can lead to diseases, such as osteomyelitis, skin, and lung infections in humans. Murine infection models have been an essential tool to the study of S. aureus virulence, although they do have their limitations. In murine pneumonia models, S. aureus infections are generally not well maintained. We hypothesize that culturing S. aureus under different conditions, such as in Synthetic CF Medium 2 (SCFM2), would increase bacterial respiratory loads compared to growing the bacteria on standard rich medium agar plates. We observed that culturing six S. aureus strains from various sources in SCFM2 resulted in a neutral or increased level of bacterial lung load compared with those cultured on agar plates. Focusing on one strain, WU1, we found that culturing in SCFM2 improved bacterial persistence in the oropharynx and led to a sustained long-term lung infection compared with agar plates. We further noted that mice experienced increased inflammation in both lobes of the lungs when infected with WU1 cultured in SCFM2 compared with agar plates. Overall, we have shown that culturing S. aureus under different conditions prior to infection impacts the level of colonization and host response.

Pasteurella multocida causes several severe animal diseases, including hemorrhagic septicemia in ungulates. Strains of P. multocida are differentiated into five capsular and nine lipopolysaccharide genotypes. To date, only P. multocida strains that produce capsule types B and E have been recovered from cases of hemorrhagic septicemia. The structure of the type B capsule has recently been determined to be a repeating main chain of N-acetyl glucosamine (GlcNAc) and N-acetyl mannosaminuronic acid (ManNAcA), with a fructose and glycine side chain. In this study, we investigated the role of proteins involved in capsule biosynthesis in P. multocida strain M1404, a bison hemorrhagic septicemia isolate that produces a type B capsule. Using capsule absorbance assays, Alcian blue stains, and capsule Western blots to assess capsule production and export, as well as in-source collision-induced dissociation mass spectrometry to determine capsule structure, we show that BcbC is likely the capsule synthase; BcbE and BcbG are involved in fructose side chain addition, and BcbH is likely the glycine transferase. Furthermore, we show that inactivation of bcbE, bcbG, or bcbH significantly decreases overall capsule production in P. multocida strain M1404. Additionally, heterologous expression of bcbABCDI in a P. multocida strain VP161 (type A) capsule mutant allowed production of a repeating GlcNAc-ManNAcA monomer in this strain background that was indistinguishable from the type B main chain, indicating that these genes are necessary and sufficient for synthesis of the main repeating type B polysaccharide.

The outcome of a Plasmodium infection depends on the timely regulation of the robust pro-inflammatory response required to eliminate the parasite, but this response can cause tissue damage if not properly controlled. Interleukin-10 (IL-10) is an important regulatory cytokine that prevents immunopathology during many Plasmodium infections; however, this protection comes at the expense of less effective parasite control. This is illustrated by infection with blood-stage Plasmodium yoelii, in which mice exhibit a lower parasite load in the absence of IL-10. However, the immune components that limit parasite burden in the absence of IL-10 remain poorly understood. Abolishing IL-10 led to a predicted increase in T_H1 polarization and a higher production of IL-12 and interferon-γ (IFN-γ). However, the enhanced production of these cytokines did not explain the improved parasite control seen in Il10^-/- mice. Loss of IL-10 signaling reduced the accumulation of germinal center B cells and plasmablasts in the spleen, indicating a role for IL-10 in supporting the humoral response. However, although B cells are essential for survival, they do not play a critical role in early parasite control in IL-10-deficient mice. Moreover, Il10^-/- mice lacking IFN-γ and B cells can limit early parasite expansion, suggesting that IL-10 suppresses host-protective pathways beyond the functions of B cells and IFN-γ in parasite control.

Innate immune signaling plays a key role in host response to infection, yet the pattern recognition receptors that detect non-model gut-associated yeasts remain poorly defined. Here, we investigated macrophage sensing of Debaryomyces hansenii, a food-derived yeast that we found to be enriched within intestinal ulcers of Crohn disease (CD) patients. Using a cell surface receptor antibody screen of bone marrow-derived macrophages infected with a CD patient isolate of D. hansenii, we showed that D. hansenii-induced macrophage activation characterized by increased expression of co-stimulatory molecules, MHC-II, and pattern recognition receptors, including the C-type lectin receptor Dectin-1. Antibody blockade experiments showed both Dectin-1 and complement receptor 3 subunit CD11b were required for phagocytosis of D. hansenii, while Dectin-1 was uniquely required for production of the pro-inflammatory cytokine tumor necrosis factor (Tnf). CRISPR-Cas9-mediated deletion of Dectin-1 phenocopied antibody neutralization effects on phagocytosis. Furthermore, deletion of Dectin-1 or its downstream signaling adaptor molecule Card9 resulted in reduced Tnf secretion in response to D. hansenii. Dectin-1-mediated uptake of D. hansenii was observed in primary bone marrow-derived macrophage and dendritic cells, as well as across the spectrum of macrophage polarization states. Together, these findings define the role of Dectin-1-Card9 signaling axis in innate immune cell sensing of D. hansenii. These findings support the emerging relevance of innate immune recognition of a yeast in Crohn disease pathogenesis.

Pseudomonas aeruginosa (Pa) is a ubiquitous, opportunistic nosocomial pathogen that poses a significant threat due to its innate and acquired multidrug resistance. Novel vaccine strategies are urgently needed for vulnerable populations, many of which harbor pre-existing immunity from prior encounters with Pa. Here, we evaluated a multivalent subunit vaccine combining type III secretion system (T3SS) antigens and exolysin A (ExlA) in a nanoemulsion formulation using a clinically relevant murine pulmonary pre-exposure model. This approach allowed us to determine whether vaccination could overcome the limitations of the host's initial, ineffective immune response. Vaccination fundamentally transforms suboptimal baseline memory into a potent, multi-faceted Th1/Th17-polarized response. This globally transformed signature was characterized by significantly enhanced antigen-specific IFN-γ and IL-17A production, both locally and systematically in the lung, with exceptionally large biological effect sizes (Cohen's d values reaching 21.35). By utilizing log10 transformation to accurately reflect pathogen growth kinetics, we demonstrated that this vaccine-augmented immunity conferred statistically significant protection following heterologous challenge. In the twice-exposed cohort, vaccination promoted superior bacterial clearance of the T3SS-positive strain, though clearance of the ExlA-positive strain was not enhanced, potentially due to immunological interference from pre-existing T3SS memory. In the thrice-exposed cohort, a functional protective threshold was observed, where high levels of natural immunity matched the vaccine-induced clearance levels. Our findings established that our vaccine formulation can effectively boost and redirect pre-existing immunity, offering a promising approach to overcome the limitations of natural exposure and protect at-risk individuals from diverse Pa infections.