Infection and Immunity最新文献

Tuberculosis (TB) is notoriously difficult to treat, likely due to the complex host-pathogen interactions driven by pathogen heterogeneity. An understudied area of TB pathogenesis is host responses to Mycobacterium tuberculosis bacteria (Mtb) that are limited in zinc ions. This distinct population resides in necrotic granulomas and sputum and could be the key player in tuberculosis pathogenicity. In this study, we tested the hypothesis that macrophages differentiate between Mtb grown under zinc limitation or in the standard zinc-replete medium. Using several macrophage infection models, such as murine RAW 264.7 and murine bone marrow-derived macrophages (BMDMs), as well as human THP-1-derived macrophages, we show that macrophages infected with zinc-limited Mtb have increased bacterial burden compared with macrophages infected with zinc-replete Mtb. We further demonstrate that macrophage infection with zinc-limited Mtb trigger higher production of reactive oxygen species (ROS) and cause more macrophage death. Furthermore, the increased ROS production is linked to the increased phagocytosis of zinc-limited Mtb, whereas cell death is not. Finally, transcriptional analysis of RAW 264.7 macrophages demonstrates that macrophages have more robust pro-inflammatory responses when infected with zinc-limited Mtb than zinc-replete Mtb. Together, our findings suggest that Mtb's access to zinc affects their interaction with macrophages and that zinc-limited Mtb may be influencing TB progression. Therefore, zinc availability in bacterial growth medium should be considered in TB drug and vaccine developments.

Over the last decade, a Neisseria meningitidis (Nm) urethritis-causing clade (NmUC) has emerged to cause clusters of meningococcal urethritis in the United States and globally. One genomic signature of NmUC is the integration of Neisseria gonorrhoeae (Ng) DNA in an operon, NEIS1446-NEIS1438, which partially replaced the Nm ispD gene. IspD is the 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase of the terpenoid precursor synthesis pathway, required for the production of ubiquinones of the electron transfer chain. IspD is essential in several gram-negative bacteria. The biological importance of the NEIS1446-ispD gene conversion event for NmUC was investigated. The ispD gene was found to be essential in NmUC (CNM3) and non-clade Nm (MC58), and a mutation at the native locus can only be made with the insertion of a second ispD copy in the genome. The IspD_MC58 variant was more efficient at promoting aerobic growth at a low level than IspD_CNM3; the two proteins differ by 15 residues. Maximal aerobic growth densities of strains with an NmUC background resembled Ng (FA19), and both were significantly lower than Nm. In contrast to non-clade Nm, all NmUC strains survived well anaerobically. Increasing ispD expression by titrating IPTG in non-clade Nm enhanced anaerobic survival. Translational reporters of the NmUC and Ng promoters demonstrated similar expression levels, and both were significantly higher than non-clade Nm, under aerobic and microaerobic conditions. Our findings suggest that the integration of gonococcal DNA into the NEIS1446-NEIS1438 operon of NmUC has increased ispD expression, contributing to NmUC's adaptation to the oxygen-limited environment of the human urogenital tract.

Hypervirulent Klebsiella pneumoniae (hvKp) strains are considered to be relatively rare in the United States, but cases are increasingly reported. We prospectively and serially collected K. pneumoniae clinical isolates identified in respiratory specimens at a health system in Western Pennsylvania between 2020 and 2022. A total of 273 K. pneumoniae isolates from 216 unique patients were analyzed for markers of hypervirulence by both string test for a hypermucoid phenotype and multiplex PCR to detect isolates carrying cardinal virulence genes rmpA, rmpA2, iutA, and iro. Of the 273 isolates, 13 (4.8%) tested positive by string test including 11 nonduplicate K. pneumoniae isolates, and two of these (0.7%) were positive by PCR for virulence genes rmpA, rmpA2, iutA, and iro. The latter two putative hvKp strains, belonging to sequence types ST23-K1 and ST86-SLV-K2, possessed pLVPK-like plasmids, and were collected from community-associated infections in individuals without known travel histories. Both putative hvKp strains and two additional string test-positive strains were resistant to killing by human serum. The hvKp strains caused significant pneumonia in mice infected by oropharyngeal aspiration, with significantly higher weight loss and increased bacterial burden in the lungs of mice infected with the KL1 (ST23) strain compared to the KL2 (ST86-SLV) strain. We also observed decreased survival of mice infected with the KL1 strain compared to the KL2 strain. These findings add to the growing body of evidence suggesting that hvKp strains, once considered endemic to Asia, may now be circulating in North America.IMPORTANCECertain lineages of Klebsiella pneumoniae are increasingly recognized to cause severe community-associated infection, but information on their prevalence in the United States is limited. In a prospective, sequential cohort of 273 K. pneumoniae respiratory isolates, we identified two of them as genetically defined hypervirulent K. pneumoniae. The isolates were from local residents who developed community-onset pneumonia, suggesting that hypervirulent K. pneumoniae may already be present in the community.

Shigella infection poses a significant public health challenge in the developing world. However, lack of a widely available mouse model that replicates human shigellosis creates a major bottleneck to better understanding of disease pathogenesis and development of newer drugs and vaccines. BALB/c mice pre-treated with streptomycin and iron (FeCl₃) plus desferrioxamine intraperitoneally followed by oral infection with virulent Shigella flexneri 2a resulted in diarrhea, loss of body weight, bacterial colonization and progressive colitis characterized by disruption of epithelial lining, loss of crypt architecture with goblet cell depletion, increased polymorphonuclear infiltration into the mucosa, submucosal swelling (edema), and raised proinflammatory cytokines and chemokines in the large intestine. To evaluate the usefulness of the model for vaccine efficacy studies, mice were immunized intranasally with a recombinant protein vaccine containing Shigella invasion protein invasion plasmid antigen B (IpaB). Vaccinated mice conferred protection against Shigella, indicating that the model is suitable for testing of vaccine candidates. To protect both Shigella and Salmonella, a chimeric recombinant vaccine (rIpaB-T2544) was developed by fusing IpaB with Salmonella outer membrane protein T2544. Vaccinated mice developed antigen-specific serum IgG and IgA antibodies and a balanced Th1/Th2 response and were protected against oral challenge with Shigella (S. flexneri 2a, Shigella dysenteriae, and Shigella sonnei) using our present mouse model and Salmonella (Salmonella Typhi and Paratyphi) using an iron overload mouse model. We describe here the development of an oral Shigella infection model in wild-type mouse. This model was successfully used to demonstrate the immunogenicity and protective efficacy of a candidate protein subunit vaccine against Shigella.

Serotype M28 isolates of the bacterial pathogen the group A Streptococcus (GAS; Streptococcus pyogenes), but not isolates of other serotypes, have a nonrandom association with cases of puerperal sepsis, a life-threatening infection that can occur in women following childbirth. In prior studies, we established that RD2, a pathogenicity island present in all M28 GAS isolates but mostly absent from other serotypes, is a factor in the M28-puerperal sepsis association. Here, we identified a significant reduction in the RD2 conjugation frequency in inter-serotype conjugation assays relative to intra-serotype assays. As isolates of most GAS serotypes produce an antiphagocytic hyaluronic acid capsule, while M28 isolates do not, we tested whether the capsule served as a barrier to RD2 acquisition or maintenance. The data showed that capsule production had no impact on the RD2 conjugation frequency or on the ability of RD2 to enhance vaginal colonization by GAS, but did inhibit the ability of RD2 to enhance GAS adherence to vaginal epithelial cell lines. Further molecular explanations for the inter-serotype barrier to RD2 conjugative transfer were investigated, and a conserved, chromosomally encoded Type I restriction-modification system was identified as being key. We also identified that RD2 modifies the GAS transcriptome, including mRNAs encoding virulence factors with adherence and dissemination roles, following exposure to human plasma. Our data provide insights into factors that contribute to the restriction of the RD2 pathogenicity island to discrete subsets of the GAS population.

Borrelia burgdorferi, the Lyme disease pathogen, continuously changes its gene expression profile in order to adapt to ticks and mammalian hosts. The alternative sigma factor RpoS plays a central role in borrelial host adaptation. Global transcriptome analyses suggested that more than 100 genes might be regulated by RpoS, but the main part of the regulon remains unexplored. Here, we showed that the expression of bb0689, a gene encoding an outer surface lipoprotein with unknown function, was activated by RpoS. By analyzing gene expression using luciferase reporter assays and quantitative reverse transcription PCR, we found that expression of bb0689 was induced by an elevated temperature, a reduced pH, and increased cell density during in vitro cultivation. The transcriptional start site and a functional promoter for gene expression were identified in the 5' regulatory region of bb0689. The promoter was responsive to environmental stimuli and influenced by RpoS. We also showed that bb0689 expression was expressed in B. burgdorferi during animal infection, suggesting the importance of this gene for infection. We further generated a bb0689 mutant and found that the infectivity of the mutant was severely attenuated in a murine infection model. Although bb0689-deficient spirochetes exhibited no defect during in vitro growth, they were defective in resistance to osmotic stress. Cis-complementation of the mutant with a wild-type copy of bb0689 fully rescued all phenotypes. Collectively, these results demonstrate that the RpoS-regulated gene bb0689 is a key contributor to the optimal infection of B. burgdorferi in animals.

Protection against infections with intracellular bacteria requires the interaction of macrophages and T-lymphocytes, including CD8⁺ T cells. Recently, the expression of natural killer cell receptors NKG2A and NKG2C was introduced as markers of CD8⁺ T-cell subsets. The goal of this study was to functionally characterize human NKG2A and NKG2C-expressing T cells using the major pathogen Mycobacterium tuberculosis (Mtb) as a model organism. Sorted NKG2 populations were analyzed for their capacity to proliferate and degranulate and their intracellular expression of cytotoxic molecules. Cytokine release and the effect on bacterial growth were assessed after coculture of NKG2 populations with Mtb-infected macrophages. NKG2A⁺ T cells released higher levels of IFN-γ and IL-10, whereas NKG2C⁺ T cells released higher levels of IL-2, contained the greatest reservoir of intracellular granzyme B and showed a remarkable constitutive level of degranulation. Both subsets inhibited the intracellular growth of Mtb more efficiently than NKG2-negative CD8⁺ T cells. Antimicrobial activity of NKG2⁺ T cells was not associated with the release of cytokines or cytotoxic molecules. However, the frequency of polycytotoxic T cells (P-CTL), defined as CD8⁺ T cells co-expressing granzyme B, perforin, and granulysin, positively correlated with the ability of NKG2-expressing T cells to control Mtb-growth in macrophages. Our results highlight the potential of NKG2-expressing P-CTL to trigger the antibacterial activity of human macrophages. Targeting this population by preventive or therapeutic immune interventions could provide a novel strategy to combat severe infectious diseases such as tuberculosis.

In contrast to adaptive immunity, which relies on memory T and B cells for long-term pathogen-specific responses, trained immunity involves the enhancement of innate immune responses through cellular reprogramming. Experimental evidence from animal models and human studies supports the concept of trained immunity and its potential therapeutic applications in the development of personalized medicine. However, there remains a huge gap in understanding the mechanisms, identifying specific microbial triggers responsible for the induction of trained immunity. This underscores the importance of investigating the potential role of trained immunity in redefining host defense and highlights future research directions. This minireview will provide a comprehensive summary of the new paradigm of trained immunity or innate memory pathways. It will shed light on infection-induced pathways through non-specific stimulation within macrophages and natural killer cells, which will be further elaborated in multiple disease perspectives caused by infectious agents such as bacteria, fungi, and viruses. The article further elaborates on the biochemical and cellular basis of trained immunity and its impact on disease status during recurrent exposures. The review concludes with a perspective segment discussing potential therapeutic benefits, limitations, and future challenges in this area of study. The review also sheds light upon potential risks involved in the induction of trained immunity.