mBio最新文献

A quarter of a century ago, Liise-anne Pirofski and Arturo Casadevall shared their concepts of microbial pathogenesis through the lens of a damage-response framework (DRF), which characterizes disease by assessing the dynamic interactions between the host and pathogen as reflected by damage as the readout. This framework has evolved to be a powerful tool for understanding the biology of complex infectious diseases, analyzing emerging and reemerging microbes, and developing therapeutic approaches to combat infections. The DRF is also frequently used to explain research at scientific meetings and to teach microbial pathogenesis to diverse learners. This mGem reviews how the DRF came to be and provides an overview of how it is used. Without a doubt, the scientific community will continue to leverage the DRF to advance research and innovate therapeutic approaches, which is especially important as new and reemerging infectious diseases threaten global health.

In March 2024, highly pathogenic H5N1 was detected in dairy cows; as of 12 December 2024, it had spread to over 800 herds in 16 states. The ongoing outbreak is a public health crisis affecting both humans and animals, as interspecies transmission has emerged as a common characteristic of this virus. As of 12 December 2024, >30 humans have been infected in the United States related to dairy cow exposure. In this mGem, we discuss transmission modalities between cows within herds, the spread of the virus between dairy farms, and exposure risks for humans. We also highlight major gaps in knowledge constituting barriers to our ability to effectively control the spread of H5N1 in dairy cows and reduce the risks to humans.

The latent human immunodeficiency virus (HIV) reservoir presents the biggest obstacle to curing HIV chronic infection. Consequently, finding novel strategies to control the HIV reservoir is critical. Natural killer (NK) cells are essential for antiviral immunity. However, the influence of NK cell subsets and their associated inhibitory or activating receptors on their cytotoxicity toward the HIV reservoir has not been fully studied. We investigated the relationship between the percentage of NK cells or NK cell subsets and the HIV reservoir. Our results indicated that the percentage of CD56^-CD16⁺ NK cells was positively associated with HIV reservoir size (i.e., HIV DNA, HIV msRNA, or HIV usRNA). Additionally, we observed that the percentage of IFN-γ⁺ NK cells was inversely related to the HIV reservoir. Furthermore, the expression of TIGIT on NK cells, particularly CD56^-CD16⁺ and CD56^dim NK cell subsets, positively correlated with the HIV reservoir. Notably, individuals with higher percentage of TIGIT⁺ NK and lower percentage of CD226⁺ NK cells exhibited larger HIV reservoir. Mechanistically, we discovered that TIGIT could inhibit the PI3K-Akt-mTOR-mTORC1 (s6k) signaling pathway to decrease the production of IFN-γ in NK cells. Importantly, inhibiting TIGIT in NK cells enhanced their ability to eliminate reactivated latently infected CD4⁺ T cells. Our experiments underscored the crucial role of NK cells in controlling the HIV reservoir and suggested that TIGIT serves as a promising target for enhancing the NK cell-mediated clearance of the HIV reservoir.

Importance: As a major barrier to human immunodeficiency virus (HIV) cure, HIV reservoir persist in viremia-suppressed infected individuals. NK cells are important antiviral cells, and their impact on reservoir has rarely been reported. We analyzed the relationship between the size of reservoir and NK cell subsets, inhibitory receptor TIGIT expression. Our analysis found that the percentage of CD56^-CD16⁺ NK cells was positively associated with HIV reservoir size. Furthermore, TIGIT expression on NK cells and CD56^-CD16⁺ NK cells or CD56^dim NK cells has a positive correlation with the HIV reservoir. TIGIT can inhibit the PI3K-Akt-mTOR-mTORC1 (s6k) signaling pathway to decrease the production of IFN-γ on NK cells. Blocking TIGIT in NK cells can enhance their ability to eliminate reactivated latently infected CD4⁺ T cells. Our study indicated that NK cells are critical to the control of the reservoir size, and TIGIT may be a target for enhancing the NK cell-mediated elimination of the reservoir.

Phages have been shown to use diverse strategies to commandeer bacterial host cell metabolism during infection. However, for many of the physiological changes in bacteria during infection, it is often unclear if they are part of a bacterial response to infection or if they are actively driven by the phage itself. Here, we identify two phage proteins that promote efficient phage replication by reprogramming host amino acid metabolism. These proteins, Eht1 and Eht2, are expressed early in the infection cycle and increase the levels of key amino acids and the arginine-derived polyamine putrescine. This provides a fitness advantage as these metabolites are important for phage replication and are often depleted during infection. We provide evidence that Eht1 and Eht2 alter the expression of bacterial host metabolic genes, and their activities may impinge on metabolism-related signaling processes. This work provides new insight into how phages ensure access to essential host resources during infection and the competitive advantage this provides.IMPORTANCEBacterial viruses, known as phages, are abundant in all environments that are inhabited by bacteria. During the infection process, phages exploit bacterial resources, resulting in notable changes to bacterial metabolism. However, precise mechanisms underlying these changes, and if they are driven by the phage or are a generalized bacterial response to infection, remain poorly understood. We characterized two proteins in Pseudomonas aeruginosa phage JBD44 whose activities alter bacterial host metabolism to optimize phage replication. Our work provides insight into how phages control bacterial processes to ensure access to essential host resources during infection.

No organism is an island: organisms of varying taxonomic complexity, including genetic variants of a single species, can coexist in particular niches, cooperating for survival while simultaneously competing for environmental resources. In recent years, synthetic biology strategies have witnessed a surge of efforts focused on creating artificial microbial communities to tackle pressing questions about the complexity of natural systems and the interactions that underpin them. These engineered ecosystems depend on the number and nature of their members, allowing complex cell communication designs to recreate and create diverse interactions of interest. Due to its experimental simplicity, the budding yeast Saccharomyces cerevisiae has been harnessed to establish a mixture of varied cell populations with the potential to explore synthetic ecology, metabolic bioprocessing, biosensing, and pattern formation. Indeed, engineered yeast communities enable advanced molecule detection dynamics and logic operations. Here, we present a concise overview of the state-of-the-art, highlighting examples that exploit optogenetics to manipulate, through light stimulation, key yeast phenotypes at the community level, with unprecedented spatial and temporal regulation. Hence, we envision a bright future where the application of optogenetic approaches in synthetic communities (optoecology) illuminates the intricate dynamics of complex ecosystems and drives innovations in metabolic engineering strategies.

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.

African swine fever virus (ASFV) is a high-consequence pathogen posing a substantial threat to global food security. This large DNA virus encodes more than 150 open reading frames, many of which are uncharacterized. The EP402R gene encodes CD2v, a glycoprotein expressed on the surface of infected cells and the only viral protein known to be present in the virus external envelope. This protein mediates binding of erythrocytes to both cells and virions. This interaction is known to prolong virus persistence in blood thus facilitating viral transmission. The sequence of the extracellular domain of CD2v shows similarity with that of mammalian CD2 proteins and is therefore likely to feature two immunoglobulin (Ig)-like domains. A combination of protein structure modeling and extensive mutagenesis was used to identify residues mediating binding of transiently expressed CD2v to erythrocytes. The N-terminal Ig-like domain AGFCC'C″ β sheet was identified as the putative CD2v erythrocyte-binding area. This region differed from the putative CD58 ligand binding site of host CD2, suggesting that CD2v may bind to a ligand(s) other than CD58. An attenuated genotype I ASFV was constructed by replacing the wild-type EP402R gene for a mutant form expressing CD2v bearing a single amino acid substitution, which abrogated the binding to erythrocytes. Pigs immunized with the recombinant virus developed early antibody and cellular responses, low levels of viremia, mild clinical signs post-immunization, and high levels of protection against challenge. These findings improve our understanding of virus-host interactions and provide a promising approach to modified live vaccine development.

Importance: A better understanding of the interactions between viruses and their hosts is a crucial step in the development of strategies for controlling viral diseases, such as vaccines and antivirals. African swine fever, a pig disease with fatality rates approaching 100%, causes very substantial economic losses in affected countries, and new control measures are clearly needed. In this study, we characterized the interaction between the ASFV CD2v protein and host erythrocytes. The interaction plays a key role in viral persistence in blood since it can allow the virus to "hide" from the host immune system. We identified the amino acids in the viral protein that mediate the interaction with erythrocytes and used this information to construct a mutant virus that is no longer able to bind these cells. This virus induces strong immune responses that provide high levels of protection against infection with the deadly parental virus.

Trypanosomes have different ways of communicating with each other. While communication via quorum sensing, or by the release and uptake of extracellular vesicles, is widespread in nature, the phenomenon of flagellar fusion has only been observed in Trypanosoma brucei. We showed previously that a small proportion of procyclic culture forms (corresponding to insect midgut forms) can fuse their flagella and exchange cytosolic and membrane proteins. This happens reproducibly in cell culture. It was not known, however, if flagellar fusion also occurs in the tsetse fly host, and at what stage of the life cycle. We have developed a split-Cre-Lox system to permanently label trypanosomes that undergo flagellar fusion. Specifically, we engineered trypanosomes to contain a GFP gene flanked by Lox sites in the reverse orientation to the promoter. In addition, the cells expressed inactive halves of the Cre recombinase, either N-terminal Cre residues 1-244 (N-Cre) or C-terminal Cre residues 245-343 (C-Cre). Upon flagellar fusion, these Cre halves were exchanged between trypanosomes, forming functional full Cre and flipping reverse-GFP into its forward orientation. We showed that cells that acquired the second half Cre through flagellar fusion were permanently modified and that the cells and their progeny constitutively expressed GFP. When tsetse flies were co-infected with N-Cre and C-Cre cells, GFP-positive trypanosomes were observed in the midgut and proventriculus 28-34 days post-infection. These results show that flagellar fusion not only happens in culture but also during the natural life cycle of trypanosomes in their tsetse fly host.

Importance: We have established a procedure to permanently label pairs of trypanosomes that transiently fuse their flagella and exchange proteins. When this occurs, a reporter gene is permanently flipped from the "off" to the "on" position, resulting in the production of green fluorescent protein. Crucially, green trypanosomes can be detected in tsetse flies co-infected with the two cell lines, proving that flagellar fusion occurs in the host. To our knowledge, we are the first to describe a split-Cre-Lox system for lineage tracing and selection in trypanosomes. In addition to its use in trypanosomes, this system could be adapted for other parasites and in other contexts. For example, it could be used to determine whether flagellar fusion occurs in related parasites such as Leishmania and Trypanosoma cruzi or to monitor whether intracellular parasites and their hosts exchange proteins.

Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, we show that two different MgmA homologs are able to methylate MCR in vivo when expressed in Methanosarcina acetivorans, an organism that does not normally possess this PTM. M. acetivorans strains expressing MgmA showed small, but significant, reductions in growth rates and yields on methylotrophic substrates. Structural characterization of the Ni(II) form of Gln-methylated M. acetivorans MCR revealed no significant differences in the protein fold between the modified and unmodified enzyme; however, the purified enzyme contained the heterodisulfide reaction product, as opposed to the free cofactors found in eight prior M. acetivorans MCR structures, suggesting that substrate/product binding is altered in the modified enzyme. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation.IMPORTANCEMethane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies.

Bacterial infections can induce exuberant immune responses that can damage host tissues. Previously, we demonstrated that systemic Escherichia coli infection in mice causes tissue damage in the liver. This liver necrosis is associated with the expression of endogenous retroviruses, chromosomally integrated retroviruses that encode a reverse transcriptase. Furthermore, nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs) completely prevent tissue damage and subsequent bacterial growth within necrotic lesions. Since liver necrosis is linked to heightened systemic inflammatory responses, we hypothesized that NRTIs diminish inflammation caused by E. coli infection and may also have broad impacts on the systemic immune response to bacterial pathogens. Here, we tested this hypothesis by characterizing the effects of NRTIs on the innate immune response to bacteria. In the liver, NRTI administration following E. coli inoculation reduced the expression of a large repertoire of proinflammatory transcripts. NRTIs also had systemic anti-inflammatory effects, including reducing proinflammatory cytokine levels in serum in response to E. coli in different mouse strains. The anti-inflammatory effects of NRTIs were also apparent in response to lipopolysaccharide (LPS) and Staphylococcus aureus, suggesting that the molecular mechanisms underlying the immunomodulatory functions of NRTIs are likely conserved across distinct immune signaling pathways. Moreover, in a model of lethal LPS shock, NRTI administration prevented hypothermia and death. Together, our observations reveal that NRTIs can potently impede systemic inflammatory responses during Gram-positive and Gram-negative bacterial infections. Our findings lay the groundwork for further investigation of the therapeutic scope of NRTIs and the mechanisms underlying their anti-inflammatory effects across non-retroviral infectious diseases.IMPORTANCEInflammatory responses are critical for host control of bacterial infection, but excessive inflammation can damage host tissues and lead to sepsis. Understanding how innate immune responses are controlled during infection is important for developing new approaches to dampen excessive inflammation. In previous work, we found that tissue damage caused by excessive inflammatory responses may be driven by endogenous reverse transcriptases. Here we demonstrate that treatment of mice with reverse transcriptase inhibitors leads to broad reductions in systemic proinflammatory responses during bacterial infections and can protect mice from acute death in a lethal model of sepsis. Our findings indicate that uncovering the mechanisms underlying the anti-inflammatory functions of reverse transcriptase inhibitors may lead to new therapeutics for bacterial infectious diseases.