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The chick embryo chorioallantoic membrane (CAM) tumor model is a valuable preclinical model for studying the tumor-colonizing process of Salmonella enterica serovar Typhimurium. It offers advantages such as cost-effectiveness, rapid turnaround, reduced engraftment issues, and ease of observation. In this study, we explored and validated the applicability of the partially immune-deficient CAM tumor model. Herein, we demonstrate that Salmonella preferentially colonizes tumors and directly causes tumor cell death. Bacterial migration, tumor colonization, and intra-tumor distribution did not require flagellar-mediated motility. The vast majority of Salmonella that colonized the CAM tumor were extracellular. Thus, tumor invasion was independent of both Salmonella pathogenicity island-1-encoded and Salmonella pathogenicity island-2-encoded type III secretion systems. Surprisingly, the extracellular residence of Salmonella on CAM tumors did not require biofilm formation. We evaluated our wild-type parental strain compared to the attenuated clinical strain VNP20009 and discovered a reduced tumor colonization capability of VNP20009. The inability to effectively colonize CAM tumors potentially explains the reduced anti-tumor efficacy of VNP20009. Our work establishes the xenograft CAM model as an informative and predictive screening platform for studying tumor-colonizing Salmonella.IMPORTANCECancer has a major impact on society, as it poses a significant health burden to human populations worldwide. Salmonella Typhimurium has demonstrated promise in cancer treatment by exerting direct tumoricidal effects and enhancing host-mediated anti-tumor immunity in xenograft mouse studies. A general understanding of its pathogenesis and the relative ease of genetic manipulation support the development of attenuated strains for therapeutic use. Alternative in ovo models, such as the chorioallantoic membrane tumor model, present a suitable screening platform to accelerate the development of therapeutic strains. It allows for rapid evaluation of Salmonella strains to assess their efficacy and potential as oncolytic agents. The present study establishes that the in ovo tumor model can be utilized as a preclinical tool for evaluating oncolytic Salmonella, bridging the gap between in vitro and in vivo screening.

Post-acute sequelae of COVID-19 involves several organs, but its basis remains poorly understood. Some infected cells in mice survive the acute infection and persist for extended periods in the respiratory tract but not in other tissues. Here, we describe two experimental models of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection to assess the effect of viral virulence on previously infected cells. Both approaches use lineage tracking of previously infected cells. In mice infected with a highly pathogenic mouse-adapted SARS-CoV-2, alveolar type 2 cells (AT2) but not alveolar type 1 (AT1) cells survived the acute infection. These cells became activated, differentiated into an AT2-to-AT1 transitional cell state (KRT8⁺ pre-alveolar type 1 transitional cell state). Additionally, nearby uninfected AT2 cells upregulated the transitional marker KRT8, thereby contributing to lung regeneration. In mice sensitized to infection by transduction with Ad5-hACE2, the infection is nonlethal, and AT1 cells survived the infection. Consequently, recovery in these mice was more rapid. Taken together, these results provide an explanation for how SARS-CoV-2 virulence contributes to poor outcomes and affects clinical recovery and lung regeneration. We also identified a new mechanism by which SARS-CoV-2 impacts lung recovery, even at times when infectious virus cannot be detected.

Importance: A major consequence of the COVID-19 pandemic is that many survivors have long-term sequelae, which are not well understood. These involve many organs, with the respiratory tract being a common site of long-term effects. Many of these sequelae can be found in mice infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In this study, we have focused on the lungs, with particular interest in the fate and role of cells that were infected with SARS-CoV-2 and survived the acute infection. We found that some infected cells survive acute SARS-CoV-2 infection and that these surviving cells both contribute to the immune response in the lungs and are involved in lung recovery. These findings illustrate previously unexplored aspects of recovery from SARS-CoV-2 induced pneumonia and may be relevant for understanding aspects of post-acute sequelae of COVID-19.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing a context for future vaccine trials and emphasizing the need for further characterization of protective responses toward conserved epitopes. (201 words)IMPORTANCESeasonal influenza viruses cause hundreds of thousands of deaths each year and up to a billion infections; under the proper circumstances, influenza A viruses with pandemic potential could threaten the lives of millions more. The variable efficacies of traditional influenza virus vaccines and the desire to prevent pandemic influenzas have motivated work toward finding a universal flu vaccine. Many promising universal flu vaccine candidates currently focus on guiding immune responses to highly conserved epitopes on the central stem of the influenza hemagglutinin viral fusion protein. To support the further development of these stem-targeting vaccine candidates, in this study, we use negative stain electron microscopy to assess the prevalence of central stem-targeting antibodies in individuals who were exposed to influenza antigens through traditional vaccination and/or natural infection during the 2018-2019 flu season.

Binding to cellular receptors initiates viral replication and dictates sites in the host infected by the virus. As illustrated by mammalian orthoreovirus (reovirus), viruses can bind several types of receptors using distinct capsid components to facilitate the viral entry steps of attachment, internalization, and disassembly. The outer of the two concentric capsids of reovirus virions is formed by four viral proteins, three of which bind receptors. These capsid-receptor interactions mediate stepwise entry of reovirus, dictate viral tropism in infected animals, and expand the viral host range. Engagement of independent receptors by different capsid proteins is a property of many pathogenic viruses and illustrates common themes of receptor use in viral entry and disease.

The field of secondary metabolism has greatly benefitted from computational advances in recent years. This has been particularly true for fungal natural product studies. Strides in genome mining have led to the identification of an extraordinary number of secondary metabolite biosynthetic gene clusters (BGCs) across the fungal Kingdom and metabologenomic platforms can group BGCs into gene cluster families and link them to initial chemical structures. Missing are computational applications focused on identifying BGC regulatory networks. Here, we applied the new online gene regulatory network resource, GRAsp (Gene Regulation of Aspergillus fumigatus), to identify unknown and unpredictable BGC trans-acting transcriptional/metabolite production modules. GRAsp correctly predicted a two-component regulatory module composed of the transcription factors (TFs), RogA (regulation of gliotoxin) and HsfA, which negatively regulate the gliotoxin BGC and are also involved in gliotoxin self-protection. RogA functions through the repression of gliZ, the pathway-specific gliotoxin TF, and HsfA functions by activating rogA expression. Furthermore, GRAsp identified TFs that regulate the production of two BGCs lacking pathway-specific TFs, the helvolic acid and fumitremorgin BGCs, respectively. Finally, the known TF, NsdD, was predicted and found to regulate the hexadehydroastechrome BGC. These advances highlight the power of inference algorithms to uncover unpredictable networks in specialized metabolite synthesis.IMPORTANCEToxic secondary metabolites are virulence factors of the opportunistic fungal pathogen Aspergillus fumigatus, yet the transcriptional networks regulating secondary metabolite production remain elusive. Uncovering novel regulators without any prior information is challenging. Computational programs have gained prominence in the field of secondary metabolite research due to their accuracy and ability to handle vast amounts of data, including DNA, RNA, and protein data. In this study, a newly developed online computer platform, Gene Regulation of A. fumigatus, was used to identify five regulators involved in the production of several A. fumigatus toxins, including gliotoxin, helvolic acid, fumitremorgin, and hexadehydroastechrome. This work illustrates the potential for discovering new trans-acting regulators and mechanisms of secondary metabolite regulation through the examination of computational gene regulatory networks.

Genomic diversity in prokaryotic species is largely due to the existence of extensive pangenomes, allowing different gene complements to be drawn depending on the strain. Here, we have studied the diversity of the O-chain polysaccharide biosynthesis cluster (OBC) in marine bacteria of the Pelagibacterales order as a proxy to measure such genetic diversity in a single population. The study of single-amplified genomes (SAGs) from the whole order found a pattern similar to that of other well-studied microbes, such as the Enterobacteriales or Alteromonas, where distinct OBCs represent strains containing different gene pools. We found that most of the OBC sharing happened among individuals of the same clonal frame (>99% average nucleotide identity). Moreover, given the parsimonious way this cluster changes, the diversity of the OBCs can be extrapolated to the size of the population's pangenome. This assumes that different OBCs correspond to lineages containing unique flexible gene pools, as seen in the aforementioned microbes. Through long-read metagenomics, we could detect 380 different OBCs at a single Mediterranean sampling site. Within a single population (single species and sample) of the endemic Ia.3/VII (gMED) genomospecies, we identified 158 OBCs, of which 130 were unique. These findings suggest that the gene pool within a single population might be substantial (several thousands). While this figure is large, it aligns with the complexity of the dissolved organic matter that these organisms can potentially degrade.IMPORTANCEDifferent strains of the same bacterial species contain very different gene pools. This has been long known by epidemiologists. However, it is unknown what gene pool is present in a single set of environmental conditions, i.e., the same time and place in free-living bacteria. Here, we have leveraged information from SAGs to analyze the diversity of the gene cluster coding for the O-chain polysaccharide, a typical component of the flexible gene pool classically used as a tool to differentiate strains in clinical microbiology. It evolves at a similar rate to the rest of the genome and does not seem to be affected by an arms race with phages. One single species of Pelagibacteriales (gMED) revealed an astounding diversity in one sample studied by long-read metagenomics. Our results point to a large gene pool (local pangenome) present in a single population, which is critical to interpreting the biological meaning of the pangenome, i.e., it provides intrapopulation diversity rather than characterizing strains with different distribution in time and/or space.

Listeria monocytogenes, a foodborne pathogen, has the ability to invade intestinal mucosal cells, undergo intracellular proliferation, activate host immune responses, and induce diseases such as colitis. We have demonstrated that sentrin-specific protease 1 (SENP1) functions as a protective gene in the host, suppressing the inflammatory response triggered by Listeria monocytogenes. The host's SENP1-SIRT3 axis plays a critical role in regulating inflammation during Listeria monocytogenes infection. Our findings reveal that overexpression of SENP1, particularly under Listeria monocytogenes infection conditions (MOI = 20), effectively suppresses inflammation through modulation of glycolysis. Mechanistically, during Listeria monocytogenes infection, SENP1 accumulates in the mitochondria, facilitating the de-SUMOylation and activation of sirtuin 3 (SIRT3). Activated SIRT3 then regulates the deacetylation of pyruvate kinase M2 (PKM2), leading to a decrease in glycolytic intermediates, downregulation of glycolysis-related gene expression, and suppression of inflammation. Taken together, our study provides a deeper understanding of the mechanistic role of the SENP1-SIRT3 axis in the regulation of inflammation, offering novel insights, and strategies for the treatment and prevention of inflammatory diseases.

Importance: Sentrin-specific protease 1 (SENP1)-sirtuin 3 (SIRT3) has never been reported in the regulation of bacteria-induced inflammation. Our study demonstrated that SENP1 acted as a protective factor against Listeria-induced inflammation by promoting SIRT3 activation and subsequent metabolic reprogramming. The SENP1-SIRT3 axis served not only as an essential signaling pathway for regulating mitochondrial metabolic responses to metabolic stress but also responds to bacterial invasion and plays a protective role in the organism. Our findings provide a basis for further research into targeting the SENP1-SIRT3 signaling pathway for the treatment of bacterial infections.

Cell surface proteins determine how cells interact with their biotic and abiotic environments. In social myxobacteria, a C-terminal protein sorting tag called MYXO-CTERM is universally found within the Myxococcota phylum, where their genomes typically contain dozens of proteins with this motif. MYXO-CTERM harbors a tripartite architecture: a short signature motif containing an invariant cysteine, followed by a transmembrane helix and a short arginine-rich C-terminal region localized in the cytoplasm. In Myxococcus xanthus, MYXO-CTERM is predicted to be posttranslationally lipidated and cleaved for subsequent cell surface localization by the type II secretion system. Here, following our bioinformatic discovery, we experimentally show that myxosortase (MrtX, MXAN_2755) is responsible for the C-terminal cleavage and cell surface anchoring of TraA, a prototypic cell surface receptor. The cleavage by MrtX depends on conserved cysteines within the MYXO-CTERM motif of TraA. M. xanthus mutants lacking myxosortase are defective in TraA-mediated outer membrane exchange and exhibit cell envelope defects. In a heterologous Escherichia coli expression system, the MYXO-CTERM motif is cleaved when MrtX is co-expressed. Therefore, MrtX represents a new family of sorting enzyme that enables cell surface localization of MYXO-CTERM proteins.IMPORTANCEThe CPBP (CaaX protease and bacteriocin processing) protease family is widespread across the three domains of life. Despite considerable research on eukaryotic homologs, prokaryotic CPBP family members remain largely unexplored. In this study, we experimentally reveal the function of a novel CPBP protease called myxosortase. Our findings show that myxosortase is responsible for the C-terminal cleavage and cell surface anchoring of substrate proteins containing MYXO-CTERM motifs in Myxococcus xanthus. MYXO-CTERM cleavage also occurred in a heterologous Escherichia coli host when myxosortase is co-expressed. This is the first report that a CPBP protease is involved in protein sorting in prokaryotes. This work provides important insights into the biogenesis and anchoring of cell surface proteins in gram-negative bacteria.