PLoS Pathogens最新文献

Despite suppressive antiretroviral therapy (ART), HIV-1 persists in latent reservoirs that seed new HIV infections if ART is interrupted, necessitating lifelong therapy for people with HIV. Activation of latent HIV during ART could improve recognition and elimination of infected cells by the immune system. Protein kinase C (PKC) isozymes increase HIV transcription and hence are potential latency reversal agents. However, the clinical utility of PKCs for this application is limited due to toxicity, which is poorly understood. Our studies showed that PKC activation with multiple classes of agonists leads to widespread platelet activation, consistent with disseminated intravascular coagulation, at concentrations that were similar to those required for T-cell activation. Differential expression analysis indicated that PKC-ε and PKC-η isoforms are expressed at high levels in human CD4+ T cells but not in platelets. Using structure-based drug design, we developed a novel PKC agonist, C-233, with increased selectivity for PKC-ε. C-233 increased both supernatant HIV RNA and p24 expression ex vivo after treatment of CD4+ T cells from ART-suppressed people with HIV. C-233 was 5-fold more potent for T-cell activation relative to platelet activation. Our studies support the use of structure-based drug design to create selective novel PKC agonists for the safe activation of HIV reservoirs and improved tolerability.

A better understanding of the system-level effects of antibiotics on bacterial cells is essential to address the growing challenge of antibiotic resistance. Utilising Multipad Agarose Plate (MAP) platforms, we monitor the growth rate and cell morphology of three clinically relevant species (E.coli, S.aureus and P.aeruginosa) following exposure to 14 antibiotics across 11 concentrations (31 microbe-antibiotic combinations in total). Our results reveal a consistent increase in population growth rate heterogeneity (PGRH) as drug concentrations approach the minimum inhibitory concentration (MIC). Strikingly, the magnitude of this heterogeneity correlates with the functional distance between the ribosome and the specific cellular processes targeted by the antibiotics. Among the seven antibiotic classes studied, protein synthesis inhibitors and disruptors cause the lowest PGRH, while heterogeneity progressively increases with RNA synthesis inhibitors, DNA replication inhibitors, cell membrane disruptors and cell wall synthesis inhibitors. Because the ribosome is central to growth rate control, we hypothesize that heterogeneity might arise at the system level as a result of the propagation of damage to protein synthesis. Low heterogeneity is desirable from a clinical perspective, as high heterogeneity is often associated with persistence and treatment survival. Additionally, we observed a strong correlation between morphological alterations and growth inhibition across all antibiotics and species tested. This led to the development of a novel morphological parameter, MOR50, which enables rapid estimation of MIC for antibiotic susceptibility testing (AST) with a single snapshot after just 2.5 hours of incubation. In addition to introducing a novel, resource-efficient and rapid AST method, our findings shed new light on the system-level effects of antibiotic perturbations on bacteria, which might inform treatment design.

CCR2, a member of the G protein-coupled receptor (GPCR) superfamily, is widely expressed on monocytes, macrophages, activated T cells, and other cell types, and plays a critical role in coordinating the immune response to various infections. Here we demonstrate that CCR2 expression is significantly elevated during Chlamydia muridarum (C. muridarum) respiratory infection, and its absence leads to exacerbated susceptibility, as evidenced by significant weight loss, higher bacterial loads, severe lung pathology, and elevated levels of inflammatory cytokines (il-1β, tnfα, and il-6). The absence of ccr2 impairs both myeloid cell infiltration and T cell responses, which are crucial for effective immune defense. Specifically, ccr2 deficiency disrupts the differentiation and response of Th1 cells, which are the primary effector lineage responsible for clearing chlamydia through secretion of interferon-gamma (IFN-γ). As a result, there is a significant decrease in CD3+CD4+IFN-γ+ T cells in the lung and spleen, accompanied by reduced levels of IFN-γ protein and mRNA, as well as downregulated mRNA expression of Th1-promoting cytokines (il-12p35, il-12p40) and transcription factors (stat4, T-bet), which play crucial roles in Th1 differentiation. Moreover, ccr2 deficiency greatly diminishes STAT1 phosphorylation, a key regulator of IFN-γ secretion by Th1 cells. Meanwhile, we also observed a significant reduction in IFN-γ secretion by CD8+ T cells following ccr2 deficiency. Conversely, ccr2-/- mice exhibit an exaggerated Th2-type immune response, with elevated levels of Th2-promoting cytokines (IL-4), transcription factors (STAT6 and gata3), and il-5, which together lead to more severe lung tissue damage and increased susceptibility to infection. Furthermore, these mice show higher levels of IL-17 along with an enhanced Th17-type immune response, characterized by increased Th17-promoting cytokines TGFB, transcription factors stat3 and RORγt, and il-21, suggesting a compensatory mechanism that drives neutrophil infiltration to exacerbate lung inflammation. These findings underscore the pivotal role of CCR2, a chemokine receptor, in orchestrating the immune response to Chlamydia infection by facilitating Th1 cells differentiation while restraining Th2-type and Th17-type immune responses, thereby alleviating pulmonary inflammation.

Aspergillus fumigatus (Af) is a major mould pathogen found ubiquitously in the air. It commonly infects the airways of people with cystic fibrosis (CF) leading to Aspergillus bronchitis or allergic bronchopulmonary aspergillosis. Resident alveolar macrophages and recruited neutrophils are important first lines of defence for clearance of Af in the lung. However, their contribution to the inflammatory phenotype in CF during Af infection is not well understood. Here, utilising CFTR deficient mice we describe a hyperinflammatory phenotype in both acute and allergic murine models of pulmonary aspergillosis. We show that during aspergillosis, CFTR deficiency leads to increased alveolar macrophage death and persistent inflammation of the airways in CF, accompanied by impaired fungal control. Utilising CFTR deficient murine cells and primary human CF cells we show that at a cellular level there is increased activation of NFκB and NFAT in response to Af which, as in in vivo models, is associated with increased cell death and reduced fungal control. Taken together, these studies indicate that CFTR deficiency promotes increased activation of inflammatory pathways, the induction of macrophage cell death and reduced fungal control contributing to the hyper-inflammatory of pulmonary aspergillosis phenotypes in CF.

The var multigene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), central to host-parasite interactions. Genome structure studies have identified three major groups of var genes by specific upstream sequences (upsA, B, or C). Var with these ups groups have different chromosomal locations, transcriptional directions, and associations with disease severity. Here we explore temporal and spatial diversity of a region of var genes encoding the DBLα domain of PfEMP1 in Africa. By applying a novel ups classification algorithm (cUps) to publicly-available DBLα sequence datasets, we categorised DBLα according to association with the three ups groups, thereby avoiding the need to sequence complete genes. Data from deep sequencing of DBLα types in a local population in northern Ghana surveyed seven times from 2012 to 2017 found variants with rare-to-moderate-to-extreme frequencies, and the common variants were temporally stable in this local endemic area. Furthermore, we observed that every isolate repertoire, whether mono- or multiclonal, comprised DBLα types occurring with these frequency ranges implying a common genome structure. When comparing African countries of Ghana, Gabon, Malawi, and Uganda, we report that some DBLα types were consistently found at high frequencies in multiple African countries while others were common only at the country level. The implication of these local and pan-Africa population patterns is discussed in terms of advantage to the parasite with regards to within-host adaptation and resilience to malaria control.

To counteract hand, foot, and mouth disease-causing viruses such as enterovirus A71 and coxsackievirus A6, virus-like particles (VLPs) have emerged as a leading contender for the development of a multivalent vaccine. However, VLPs have shown rapid conversion from a highly immunogenic state to a less immunogenic state and low particle integrity lifetimes compared to inactivated virus vaccines, thus raising concerns about their overall stability. Here, we produce VLPs to investigate capsid stability using cryogenic electron microscopy (cryo-EM), mass spectrometry (MS), biochemical assays, and atomic force microscopy (AFM). In contrast to prior studies and prevailing hypotheses, we show that insect-cell produced enterovirus VLPs include encapsidated RNA fragments with viral protein coding sequences. Our integrated approach reveals that CVA6 VLPs do not undergo viral maturation, in contrast to virions; that they can encapsidate RNA fragments, similarly to virions; and that despite the latter, they are more brittle than virions. Interestingly, this indicates that CVA6 VLP stability is more affected by lack of viral maturation than the presence of RNA. Our study highlights how the development of VLPs as vaccine candidates should encompass probing for unwanted (viral) RNA content and establishing control of their maturation to enhance stability.

The ecological interplay between Streptococcus mutans and Candida albicans within dental plaque biofilms is an important factor driving pathogenesis of dental caries. This study aimed to investigate S. mutans regulation of C. albicans growth and virulence through extracellular membrane vesicles (EMVs) and modulation of ubiquitination, a key protein post-translational modification. We established a transwell co-culture model to enable "contact-independent" interactions between S. mutans and C. albicans. S. mutans EMVs were found to directly associate with C. albicans cells and promote biofilm formation and growth. Quantitative ubiquitination profiling revealed S. mutans dramatically alters the ubiquitination landscape in C. albicans. We identified 10,661 ubiquitination sites across the C. albicans proteome and their enrichment in pathways related to translation, metabolism, and stress adaptation. Co-culture with S. mutans led to upregulation of ubiquitination on 398 proteins involved in sugar catabolism and generation of reducing power. S. mutans upregulated ubiquitination of superoxide dismutase-3 of C. albicans, inducing its degradation and heightened reactive oxygen species levels, and concomitantly stimulated C. albicans growth. Our findings elucidate EMVs and ubiquitination modulation as key mechanisms governing the S. mutans-C. albicans interplay and provide new insights into the promotion of a cariogenic oral biofilm environment. This study significantly advances understanding of the complex molecular interactions underlying dental plaque dysbiosis and caries pathogenesis.