Infection and Immunity最新文献

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), continues to pose a critical global health threat as a leading infectious cause of mortality. Therapeutic efficacy is increasingly compromised by the emergence of multidrug-resistant strains and the limitations of existing regimens, which necessitate treatment durations of six months or longer. Protein tyrosine phosphatase B from Mtb (PtpB-Mtb) has been recognized as a critical virulence factor, representing a promising target for novel antitubercular therapies due to its unique structural and functional properties. In this study, a comprehensive structure-based virtual screening approach was employed to identify novel small-molecule scaffolds with inhibitory potential against PtpB-Mtb. The ChemBridge compound library was curated and filtered for drug-like properties, followed by hierarchical molecular docking and molecular dynamics simulations to prioritize candidates with high predicted affinity and stability within the PtpB-Mtb active site. Quantum mechanical calculations further characterized the electronic properties of top hits. Recombinant PtpB-Mtb was expressed and purified to homogeneity, and in vitro enzymatic assays were performed to evaluate the inhibitory potency and selectivity of shortlisted compounds. Two derivatives bearing pyrazolo[4,3-c]pyridine and 1,4-diazepane ring nuclei demonstrated significant inhibition of PtpB-Mtb activity, exhibiting IC₅₀ values of 14.4 µM and 32.6 µM, respectively. Biolayer interferometry confirmed strong and specific binding to PtpB-Mtb, with dissociation constants (K_d) of 0.012 µM and 0.57 µM. The integrated workflow presented herein highlights the potential of these novel scaffolds as starting points for the development of selective, cell-permeable PtpB-Mtb inhibitors, offering a promising avenue for next-generation anti-tubercular drug discovery.

More than half of the people with microbiologically cured tuberculosis (TB) present with post-TB lung disease (PTLD). PTLD compromises long-term respiratory health and adds to the global burden of chronic lung diseases. Despite its prevalence, the mechanisms driving tissue damage in TB are not well understood. In this review, we discuss the global burden of PTLD, evaluate host-directed therapies as promising interventions, and highlight the C3HeB/FeJ mouse model as a powerful tool for advancing pre-clinical PTLD research.

Multiple countries have observed an alarming increase in scarlet fever cases, and invasive infections often associated with a new sublineage of Streptococcus pyogenes known as M1_UK. M1_UK strains express increased levels of the streptococcal pyrogenic exotoxin A (SpeA) superantigen, and here we compare the virulence characteristics of this sublineage with the circulating M1_global strain. We obtained contemporary Canadian M1_UK isolates, and genome sequencing revealed that some M1_UK strains had acquired additional DNAse- and superantigen-encoding prophage elements, as well as an isolate with a mutation in covS. Five S. pyogenes strains were chosen for functional experiments, including 5448 (M1_global strain), M1_UK350 (a "typical" M1_UK strain), M1_UK162 (M1_UK strain containing a mutation in the covS gene), M1_UK362_ΦSP1380.vir (M1_UK strain containing a prophage element encoding the spd1, speC, and ssa genes), and M1_UK155_Φ370.1 (M1_UK strain containing a prophage element encoding the spd1 and speC genes). Exoprotein profiles demonstrated that all M1_UK background strains had enhanced production of the SpeA superantigen relative to S. pyogenes 5448. Furthermore, strains that had acquired the additional prophage elements showed enhanced activation for human T cells, although cytotoxic activity, adhesion capacity, and DNA degradation were not detectably different. Using a "humanized" superantigen-sensitive HLA-transgenic mouse infection model, the M1_UK162 covS mutant, and both M1_UK362_ΦSP1380.vir and M1_UK155_Φ370.1 strains each demonstrated increased severity during experimental skin infection compared to 5448 and M1_UK350. These findings indicate that circulating M1_UK background strains continue to acquire additional prophage-encoded virulence factors, or hypervirulent covS mutations, and that these genetic alterations may contribute to increase severity of human infections.

Mouse models have been key to studies of tuberculosis pathogenesis and drug efficacy, but many, such as those employing BALB/c mice, fail to reproduce the full range of heterogenous microniches observed in well-structured human lesions, which feature hypoxic caseous cores of necrotic debris surrounded by infected foamy macrophages. The granuloma presents a variety of environments differing in levels of oxygen, ions, nutrients, and intra versus extracellular residence, which determine the physiological state of the infecting bacillus and its susceptibility to immune or drug control. Recently, alternative mouse strains such as C3HeB/FeJ have allowed the study of infection and treatment in the context of these varied environments but exhibit substantial inconsistency in the development of hypoxic necrotic lesions, both within and between individual mice. Building on the observation that inducible nitric oxide synthase (Nos2)-deficient mice consistently develop hypoxic necrotic lesions, we have established two simplified models with infection by the aerosol route. The first uses the slightly attenuated M. tuberculosis R1Rv strain, which produces a progressive infection that is contained at a high stable burden by an adaptive immune response. In the second model, vaccination with the attenuated ∆RD1, pantothenate auxotroph mc²6230 protects from an otherwise lethal infection with virulent Mycobacterium tuberculosis Erdman. This model reflects most contemporary tuberculosis infections, which take place in the context of a pre-existing immune response from vaccination. Both variations uniformly develop well-structured hypoxic necrotic lesions and respond poorly to chemotherapy. These refined models will be useful in studies of M. tuberculosis infection and treatment.

Recurrent vulvovaginal candidiasis (RVVC), primarily caused by the fungal pathogen Candida albicans, is a common infection affecting a significant number of women worldwide. Despite a robust inflammatory response by polymorphonuclear neutrophils (PMNs) with potent antifungal properties during symptomatic episodes, fungal clearance often fails, leading to persistent overgrowth and PMN-associated immunopathology. Studies in an established animal model demonstrated that elevated vaginal heparan sulfate (HS) interferes with PMN-C. albicans interactions, thereby impairing fungal clearance. This study investigated the presence and inhibitory effects of HS in women diagnosed with RVVC. Vaginal conditioned medium (VCM) was prepared from swab samples obtained from symptomatic VVC patients, women in asymptomatic remission, and healthy controls. Results from ELISA and immunostaining showed significantly elevated HS levels in VCM-containing vaginal secretions and epithelial cells from symptomatic women compared to those from asymptomatic and healthy controls. PMN killing assays further revealed significantly reduced antifungal activity in the presence of VCM from symptomatic women compared to asymptomatic and healthy control VCM, resulting in a significant negative correlation between vaginal HS concentrations and PMN antifungal activity. The inhibitory effect of HS was further confirmed in vitro by impaired PMN killing in control VCM spiked with purified HS, and by the restoration of PMN function following heparanase (HS lyase) treatment of both symptomatic VCM and HS-spiked controls. These findings support the results from the animal studies and provide the first clinical evidence that elevated HS in the vaginal environment contributes to PMN dysfunction, leading to persistent C. albicans colonization and VVC-associated immunopathology.

Orientia tsutsugamushi, the agent of scrub typhus, is an obligate intracellular bacterium whose atypical cell wall lacks many classical pathogen-associated molecular patterns but is enriched in neutral glycans. Its recognition by phagocytes is driven by a heat-stable ligand that triggers innate cytokine responses, yet the nature of this ligand and the receptors sensing it remain incompletely understood. While activation of innate immunity via toll-like (TLR) and nucleotide-binding and oligomerization domain-like receptors has been described, recognition via C-type lectin receptors (CLRs) has remained largely unexplored. Using a flow cytometry-based screening assay with a library of CLR-Fc fusion proteins, we demonstrate binding of Orientia to four mouse CLRs, including Mincle, Dectin-1, Langerin, and DCL-1, as well as to human DC-SIGN. Binding to Mincle was Ca²⁺-dependent, indicating carbohydrate-specific recognition. In bone marrow-derived dendritic cells (BMDC), heat-inactivated Orientia induced transcriptional upregulation of Mincle in a MyD88-dependent manner, suggesting cross talk between TLR and CLR pathways. Mincle was dispensable for TNF-α induction in BMDC stimulated with Orientia but contributed to the induction of interleukin-27 and cxcl-10 mRNA, indicating an immunomodulatory rather than a classical pro-inflammatory role. We also discuss in vivo data that demonstrated upregulation of Mincle and concomitant downregulation of Dectin-1 during Orientia infection. Together, this study identifies multiple CLRs as receptors for Orientia, highlights Mincle as a modulator of innate immunity, and suggests that Mincle-driven immunoregulation helps to shape the inflammatory environment during scrub typhus.

Streptococcus gordonii is a commensal bacterium in the oral cavity and has many surface adhesins that have been well characterized. SspA/B belongs to the Antigen I/II-like family of proteins, which are well known for their multifunctional adherence capabilities. Most AgI/II-like proteins adhere to salivary agglutinin (also known as glycoprotein 340, Gp340). In an effort to identify the putative binding site on the AgI/II-like family of proteins, we conducted structural studies to determine the V-domain of SspB. In this paper, we report the structure of SspB's V-domain in complex with a TEV-peptide that was inserted to cleave the histidine tag at the C-terminus after purification. This peptide shared sequence and structural homology with a helical region on the scavenger receptor cysteine-rich (SRCR) domain of Gp340. Our studies with the synthetic peptide PepCD1^SRCR show that it inhibits the Streptococcus mutans biofilm formation in a dose-dependent manner. A comprehensive comparative analysis of this site with the corresponding sites in the homologous V-domains of S. mutans AgI/II and GbpC established that most of these interface residues were conserved. Based on the structural data, mutational analysis was initiated to study the effect of binding-interface residues on the ability of each of these V-domains from S. mutans and S. gordonii to adhere to salivary agglutinin. Here, we report for the first time the binding site for the V-regions that are distinct among oral streptococci, which provides potential opportunities for therapeutic intervention of pathogenic species.

Cryptosporidium is a protozoan parasite that causes cryptosporidiosis, an enteric infection associated with diarrhea, malnutrition, and impaired childhood development in low- and middle-income countries. Both humoral and cell-mediated immune responses have been implicated in protection, but the durability and quality of human immune responses in immunocompetent adults remain poorly defined. We investigated the development of immunity in two healthy U.S. adults following primary cryptosporidiosis acquired during travel to Bangladesh. Longitudinal plasma samples were analyzed for antibody responses to Cryptosporidium antigens Cp17 and Cp23 and for circulating cytokine profiles. Circulating antibody peaked at 3 weeks post-infection but declined rapidly thereafter, approaching baseline within 16 weeks. In contrast, antibody avidity increased steadily over time, consistent with ongoing affinity maturation in germinal centers. While affinity maturation occurred, the composition of memory B cells specific to Cryptosporidium antigens was skewed toward IgM+ cells across time points, suggesting extrafollicular responses dominated and germinal center-derived, class-switched memory was limited. Cytokine profiling revealed an acute Th1-skewed response, with elevations in CXCL9, CXCL10, IL-27, IFNγ, IL-12, and IL-18 during early infection. These signatures mirrored protective pathways identified in murine models, underscoring the importance of type I immunity in parasite clearance. Together, these findings highlight that while antibody responses to Cryptosporidium are short-lived, avidity maturation persists, and Th1-driven cytokine responses dominate during acute infection. This work provides rare longitudinal data on immune responses in naïve adults following natural cryptosporidiosis and offers insight into mechanisms that may inform vaccine development and strategies to mitigate recurrent infection in vulnerable populations.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen and a common cause of antibiotic-resistant infections including neonatal sepsis and hospital-acquired pneumonia. K. pneumoniae strains can be categorized into two pathotypes: classical K. pneumoniae (cKp), which often causes nosocomial infections, and hypervirulent K. pneumoniae (hvKp), which can cause severe disease in healthy hosts. New therapies are urgently needed for these infections, and caseinolytic proteins have emerged as promising therapeutic targets in other bacterial pathogens. ClpX and ClpP have been implicated in bacterial protein homeostasis, regulation of virulence, and antimicrobial susceptibility in other species, but their specific roles in K. pneumoniae pathogenesis have yet to be defined. Here, we investigate the contribution of K. pneumoniae ClpX and ClpP to lung infection, virulence factor regulation, and antibiotic susceptibility. In a murine pneumonia model, loss of ClpX impairs infection of both hvKp and cKp. Loss of ClpX results in decreased capsule production in hvKp and enhances type 1 pilus production in both pathotypes. In hvKp, loss of ClpX increases type 3 pili, while in cKp, increased type 3 piliation is observed with loss of ClpP. Across both pathotypes, loss of ClpX or ClpP increases susceptibility to a range of antibiotics. These data identify ClpX as critical to K. pneumoniae virulence and antimicrobial susceptibility. By connecting ClpX to capsule production, pili regulation, and in vivo virulence, this work highlights a conserved putative therapeutic target that may enable adjunctive strategies to enhance antibiotic efficacy or attenuate the severity of K. pneumoniae infection.IMPORTANCEKlebsiella pneumoniae is a leading cause of antibiotic-resistant and hospital-acquired infections. The emergence of highly virulent strains of K. pneumoniae capable of causing severe disease is of utmost concern. Here, we investigate two specific caseinolytic proteins, ClpX and ClpP, produced by both classical and hypervirulent strains of K. pneumoniae and their role in K. pneumoniae lung infection. We show that ClpX is a key regulator of virulence factors including bacterial pili and capsule; it is essential for murine pulmonary fitness across both classical and hypervirulent pathotypes. Furthermore, loss of ClpX increases susceptibility to multiple antibiotics, indicating a role in both protein homeostasis and pathogenicity. These findings suggest ClpX is a conserved virulence determinant in multiple strains of K. pneumoniae and highlight its potential as a therapeutic target to enhance antibiotic efficacy or mitigate disease severity.

Metabolic adaptation to the host environment is a key determinant of bacterial pathogenesis, enabling both colonization and invasive disease. This is particularly true for Neisseria gonorrhoeae (Gc), the causative agent of gonorrhea, which lacks effector-injecting secretion systems or toxins. Gc infection triggers a rapid influx of neutrophils (polymorphonuclear cells [PMNs) that typically kill bacteria through multiple mechanisms, including a potent oxidative burst. Despite this, Gc exhibits remarkable resistance to reactive oxygen species and readily replicates in the presence of PMNs, which is in part due to the consumption of PMN-derived lactate. Previous studies demonstrated that the lactate permease, LctP, is required for oxidative stress resistance in Gc and host colonization in a murine model of gonorrhea, suggesting that lactate utilization contributes to virulence. Gc encodes four lactate dehydrogenases (LDHs) with distinct regulation and mechanisms, including two L-LDHs, LldD and LutACB. Although either enzyme alone supports L-lactate utilization, we found that both are required for full fitness during co-colonization with PMNs, indicating some non-redundant roles. Furthermore, LldD enhances oxidative stress resistance and is required for Gc colonization in a murine model of gonorrhea, whereas LutACB is dispensable. These findings identify LldD as a key factor promoting oxidative stress resistance, survival during PMN challenge, and host colonization.