PLoS Pathogens最新文献

Bordetella atropi is an intracellular bacterial pathogen that infects the intestinal epithelia of the nematode host Oscheius tipulae. We previously showed that the bacteria use filamentation as a novel cell-to-cell spreading mechanism once inside the intestinal cell. However, how the bacteria invade the host cells and what factors contribute to B. atropi infection process remain unknown. In this study, we investigate the roles of type III (T3SS) and type VI secretion systems (T6SS) in B. atropi pathogenesis, which are employed by many bacterial pathogens, both extracellular and intracellular, to deliver effectors that manipulate host physiology to their advantage. We found that the two T6SSs encoded in B. atropi genome played no obvious roles in the invasion or intracellular spreading. In contrast, a T3SS was required for intestinal cell invasion. T3SS mutants showed loss of host cell protrusions from the apical surface that normally engulf invading wild type bacteria, as seen by both electron microscopy and confocal fluorescent microscopy. These protrusions bear morphological similarities to membrane ruffles triggered by the T3SS-mediated invasion seen in other pathogens such as Salmonella and Shigella spp. Additionally, we conducted dual transcriptomics and saw upregulation of T3SS in vivo, along with several putative effectors and the virulence regulator BvgS of the genus Bordetellae. We knocked out these effector candidates and found that deletion of one of these genes, deiA (decreased invasion protein A), leads to a reduction in the number of invasion events and overall percentage of infected animals in the population. In addition, deletion of the virulence regulator bvgS resulted in a complete loss of B. atropi invasion, suggesting it may regulate T3SS for host cell invasion.

The World Health Organisation has identified Acinetobacter baumannii as a critical priority antimicrobial resistant (AMR) pathogen for which new therapeutics are needed. Despite this, currently there are no antibody or vaccine candidates in advanced clinical development for A. baumannii. To help address this, we designed a protein microarray approach to identify multiple A. baumannii protein antigens for further investigation as potential targets for vaccination or an antibody therapy. An 868-protein microarray was constructed containing mainly highly conserved A. baumannii proteins, and was enriched for those predicted to be surface localised and for which the corresponding gene is highly expressed during culture in ex vivo human serum. Probing the protein microarray with sera obtained from mice after non-lethal infection with multiple different A. baumannii strains identified IgG responses to 66 proteins. Four proteins (three previously poorly described outer membrane proteins and BamA, a known protective vaccine antigen selected as a positive control) were selected for further investigation. Polyclonal rabbit IgG to all four protein antigens recognised multiple clinical AMR A. baumannii strains, and for selected strains promoted opsonisation with IgG and complement, improved neutrophil phagocytosis, and increased membrane attack complex formation. Passive immunisation with polyclonal IgG to each antigen partially protected mice against A. baumannii sepsis, and a combination of polyclonal to two antigens completely protected against A. baumannii murine sepsis. Repeating passive immunisation experiments in mice depleted of complement, neutrophils or tissue macrophages demonstrated protection against systemic infection was dependent on complement and neutrophils but not macrophages. Overall, the data demonstrate that our protein microarray is a novel approach that can rapidly identify multiple new protein antigens as potential antibody targets for preventing or treating AMR bacterial infections.

Staphylococcus aureus (S. aureus)-driven senescence of bovine mammary epithelial cells is a key determinant of mammary gland health, yet its molecular basis remains poorly defined. Sirtuin 5 (SIRT5), a mitochondria-localized desuccinylase, may play an important regulatory role in this process. This study aimed to elucidate the mechanisms by which S. aureus drives cellular senescence and to define the contribution of the SIRT5-mitochondrial axis to delaying senescence. We found pronounced oxidative stress and cellular senescence in mammary tissues from cows with S. aureus mastitis, accompanied by marked downregulation of SIRT5. In an S. aureus-infected epithelial cell model, infection induced mitochondrial stress characterized by excessive mitochondrial fragmentation, loss of membrane potential, and increased mitochondrial superoxide, along with oxidative damage and cellular senescence. Mechanistically, S. aureus toxins and the toxin-induced inflammatory response cooperatively drove mitochondrial stress, which in turn increased intracellular bacterial burden and exacerbated cell death. During infection, SIRT5 protein abundance was significantly reduced. Mass spectrometry and co-immunoprecipitation analyses indicated that infection upregulated the ubiquitin-conjugating enzyme ubiquitin C (UBC), enhanced its interaction with SIRT5, and promoted ubiquitin-mediated degradation of SIRT5. Loss of SIRT5 increased succinylation of dynamin-related protein 1 (DRP1), inhibited its ubiquitin-mediated degradation, and led to its excessive accumulation on the outer mitochondrial membrane, thereby promoting excessive mitochondrial fission. Functionally, SIRT5 overexpression markedly alleviated mitochondrial stress, oxidative damage, and senescence phenotypes. When mitochondrial fission was forcibly enhanced, the cytoprotective effect of SIRT5 was substantially weakened, confirming that SIRT5 acts through a pathway dependent on mitochondrial integrity. Collectively, S. aureus infection releases toxins and induces inflammatory injury, during which UBC-mediated SIRT5 degradation activates DRP1-dependent mitochondrial hyper-fragmentation, aggravating mitochondrial stress, oxidative stress, and mammary epithelial cell senescence. These findings identify SIRT5 as a critical regulator of redox and mitochondrial homeostasis in mammary epithelial cells and a potential therapeutic target for mitigating oxidative damage associated with bovine mastitis.

The caseinolytic protease (ClpP) is an emerging antibacterial target. Pseudomonas plecoglossicida (Pp), a pathogen causing visceral white spot disease in Larimichthys crocea, encodes two ClpP paralogs, PpClpP1 and PpClpP2. This study characterizes their distinct structural and functional properties. Phylogenetic and biochemical analysis revealed that PpClpP2 functions as a canonical serine protease with high peptidase activity, while PpClpP1 is evolutionarily divergent, exhibiting low inherent activity due to an unconventional Ser-His-Pro catalytic triad and a truncated N-terminal domain. Cryo-EM structure determination of PpClpP1 confirmed a homotetradecameric assembly with a dilated axial pore and a non-canonical catalytic geometry. In contrast, AlphaFold-predicted PpClpP2 displayed a compact structure with a canonical Ser-His-Asp triad. The subunits formed a stable heterotetradecamer (PpClpP1P2) with enhanced proteolytic activity compared to individual homotetradecameric. Pull-down assays demonstrated that PpClpP2, but not PpClpP1, specifically interacts with the unfoldase PpClpX, and the PpClpP1P2 heterotetradecamer further augmented PpClpX-mediated degradation of model substrates. Notably, the proteasome inhibitor bortezomib (BTZ) selectively inhibited PpClpP1 by binding to a unique pocket near the active site without engaging the catalytic serine, thereby suppressing bacterial growth in a PpClpP1-dependent manner. This study elucidates the structural basis of functional divergence between PpClpP paralogs, highlights their synergistic interplay in proteolysis, and identifies PpClpP1 as a druggable target for antibacterial development.

Sepsis is a life-threatening condition characterized by a dysregulated immune response to infection, often leading to organ dysfunction and even death. During the recovery phase of sepsis, patients frequently exhibit impaired antimicrobial function of immune cells, which exacerbates the state of immunosuppression and increases the risk of secondary infections. However, therapeutic strategies targeting sepsis-induced immunosuppression have yet to achieve breakthrough progress, with the core challenge lying in the significant gaps in understanding the molecular mechanisms underlying immunosuppression. In this study, we integrated clinical samples, mouse models, and molecular mechanisms to reveal that the reduction in macrophage function and epigenetic dysregulation, particularly histone ubiquitination, are central drivers of sepsis-induced immunosuppression. Further investigation demonstrated that MYSM1, a deubiquitinase, plays a pivotal role in regulating this ubiquitination process. Targeted deletion of the N-terminal domain of MYSM1 markedly enhances the inflammatory response during the early phase of secondary infection in sepsis, facilitating bacterial clearance and significantly mitigating tissue damage in the late phase of secondary infection, thereby improving the survival outcomes in mice. Overall, our study elucidates the role of MYSM1-mediated dysregulation of epigenetic modifications in the immune response during the late phase of sepsis, providing a novel therapeutic approach for addressing sepsis-related immune dysfunction.

Careful regulation of type I interferons (IFN) like IFN-β is vital for balancing tissue damage and protection against infections. Heterogeneity in type I IFN expression among virally infected cells is a common phenomenon that may help limit IFN responses, but the source of this heterogeneity is poorly understood. We previously found that during Kaposi's sarcoma-associated herpesvirus replication, type I IFN induction was limited to a small percentage of infected cells. This heterogeneity was not explained by viral gene expression. Here, we used a fluorescent reporter and fluorescence activated cell sorting to investigate the source of the heterogeneity. Surprisingly, the canonical IFN induction pathway culminating in the activation of the IRF3 transcription factor was similarly activated between cells that made high vs. low/no IFN-β. In contrast, the activation or expression of the two other IFN transcription factors, the NF-κB subunit RelA and the AP-1 subunit ATF2, correlated with IFN-β induction. Our results suggest that during viral infection, activation of IRF3 does not automatically result in IFN responses at the level of individual cells, but that other factors, such as NF-κB and AP-1, are limiting for type I IFN induction.

Scrub typhus, caused by Orientia tsutsugamushi (Ot) bacteria, is a serious acute febrile illness associated with significant mortality. No effective vaccine is currently available, largely due to the complex Ot strain diversity and an incomplete understanding of protective immune mechanisms. To overcome these challenges, there is a critical need for a suitable animal model that mimics human disease through the natural route of infection. Here, we report for the first time that a genetically engineered humanized mouse strain (with triple knockout/knock-in of IFN-γ and its receptors, abbreviated as hIFNG/hIFNGR), exhibits increased susceptibility to intradermal Ot infection compared to wild-type (WT) mice. This is evidenced by greater body weight loss, elevated bacterial burden, and reduced expression of interferon-stimulated genes (ISGs). hIFNG/hIFNGR mice exhibit pronounced biochemical abnormalities and tissue pathology accompanied by dysregulated T cell and neutrophil responses following infection. Notably, this novel mouse strain with human IFN-γ signaling can develop skin eschar-like lesions resembling those observed in human patients. Overall, our study introduces a promising mouse model to dissect the immunopathogenesis of scrub typhus and evaluate future vaccine candidates.

Carbapenem-resistant (CR) organisms (CRO) have been identified as critical priority pathogens, emphasizing the urgent need for novel therapeutic strategies. Combination therapy emerges as a promising approach to address multidrug-resistant bacterial infections. Here we demonstrate that eravacycline (ERV), in combination with amikacin (AMK), effectively eliminates a panel of clinically isolated CR Escherichia coli, CR Klebsiella pneumoniae, and CR Acinetobacter baumannii. Mechanistically, the AMK-ERV combination enhances bacterial oxidative phosphorylation, leading to an accumulation of reactive oxygen species, which induce oxidative stress and accelerate bacterial cell death. Notably, this combination significantly improves survival rates in mouse models of intra-abdominal infection, demonstrating efficacy against infections induced by CR pathogens. Furthermore, serum metabolomics reveals that the AMK-ERV combination upregulates metabolic pathways of lipids and amino acids. Interestingly, the amino acid methionine significantly enhances the antibacterial activity of ERV against CR pathogens both in vitro and in vivo. Our findings underscore the potential of repurposing AMK in combination with ERV to combat CR pathogens and propose a novel strategy for controlling these infections through the combination of antibiotics with specific metabolites such as methionine.

[This corrects the article DOI: 10.1371/journal.ppat.1013054.].