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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a uniquely successful pathogen due in large part to its complex lipid-rich cell envelope. Comprising nearly 40% of its dry weight, Mtb lipids-such as mycolic acids, phthiocerol dimycocerosates (PDIM), trehalose dimycolate (TDM), and sulfolipids (SLs)-play crucial roles in infection, immune evasion, intracellular persistence, granuloma formation, transmission, and drug resistance. These lipids modulate host-pathogen interactions by altering host membrane biophysics, hijacking phagosome maturation, and interfering with host immune pathways, including autophagy and inflammatory signaling. Upon inhalation, Mtb surface lipids inhibit pulmonary surfactant function and mask pathogen-associated molecular patterns, facilitating uptake by permissive macrophage subsets. Intracellularly, lipoglycans like mannose-capped lipoarabinomannan block phagolysosome fusion, while PDIM and TDM promote phagosomal escape and subversion of vesicular trafficking. Lipid-mediated modulation of autophagy pathways further enhances bacterial survival within host cells. In addition to shaping host immune responses, Mtb lipids orchestrate granuloma development and promote pathological features such as foam cell formation and caseation, which are central to transmission. Specifically, phenolic glycolipids and SLs stimulate neuronal pathways, triggering cough, thereby facilitating aerosol spread. Finally, the lipid-rich envelope acts as a formidable barrier to antibiotics, with resistance partly driven by the altered lipid composition and architecture in multidrug-resistant strains. Targeting lipid biosynthesis and transport pathways offers promising avenues for novel anti-TB therapies. This review highlights the multifaceted roles of Mtb lipids at the host-pathogen interface, recent technical advances enabling these insights, and emerging challenges in translating lipid biology into improved TB control.

Despite the availability of prophylactic vaccines, human papillomavirus (HPV) infection remains the leading viral cause of cancer worldwide. The HPV E7 oncoprotein is a key factor in cancer progression by degrading host tumor suppressor proteins, thus offering a promising target for antiviral therapy. In this study, we screened a panel of high-affinity antibodies against HPV18 E7. Through evaluation of their cytotoxic effects in HPV18-positive HeLa cells, incorporating the structure of antibodies and the structural simulation of complexes, we identified the α3 helix of the HPV18 E7 protein and its adjacent groove as a novel and effective antiviral epitope. The candidate antibody 17F2 showed efficacy in inhibiting cell proliferation and tumor formation when transfected into HeLa cells as a single-chain variable fragment (scFv). To directly assess the efficacy of the antibody and enhance the accessibility of antibody drugs, we employed an mRNA platform for scFv delivery. This approach significantly inhibited the growth of HPV18-positive tumors in the immunodeficient mouse models. Our study identifies the α3 helix of HPV18 E7 as a viable antiviral target and provides proof of concept for mRNA-encoded scFv antibodies as a novel and effective strategy to neutralize viral oncoproteins in the treatment of HPV-related cancers.

Importance: The development of effective therapeutics against human papillomavirus (HPV)-related cancers remains an urgent medical priority. While therapeutic vaccines depend on the host immune response, their efficacy can be limited in immunocompromised individuals. In contrast, antibody-based therapies that directly target viral oncoproteins represent a promising alternative with a more immediate mechanism. In this study, we identified and characterized a potent therapeutic antibody against HPV18 E7 and uncovered an unrecognized targeting epitope within this viral oncoprotein. Moreover, we addressed a major limitation of conventional antibody therapies-their inability to efficiently target intracellular proteins-by employing an mRNA-lipid nanoparticle delivery platform for intracellular expression of the antibody. As a result, we have developed a novel candidate drug with a clear mechanism, offering a new strategy for the treatment of cancers associated with HPV18.

Bacterial virulence is regulated by the growth phase and ribosome activity, implicating the formation of translationally silent ("hibernating") ribosomes. Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular bacterium that grows in both environmental amoebae and mammalian macrophages. Thus far, ribosome hibernation factors of L. pneumophila have not been characterized. Here, we show that L. pneumophila encodes homologs of the ribosome hibernating factors LhpF (Lpg1206), RaiA (Lpg0467), RsfS (Lpg1377), and the GTPase HflX (Lpg0010), which define the ribosome populations by mediating 100S ribosome dimerization, 70S inactivation, ribosome assembly inhibition, and ribosome splitting, respectively. Exceptional among γ-proteobacteria, L. pneumophila forms 100S ribosome dimers during exponential growth. Functional studies show that the hibernation factors support survival upon starvation, regrowth, efficient host infection, and virulence factor production of L. pneumophila. Furthermore, they enhance antibiotic tolerance and shape intracellular heterogeneity of bacterial growth and motility. Our findings identify ribosome hibernation as a central mechanism by which L. pneumophila orchestrates survival, persistence, and infection, highlighting its critical role in bacterial physiology and pathogenesis.IMPORTANCEDue to nutrient limitation and adverse conditions in the environment, bacteria mostly do not grow exponentially but adopt a resting ("dormant") state. Bacterial dormancy usually coincides with the formation of translationally silent ("hibernating") ribosomes; however, the role of ribosome hibernation in intracellular pathogens is poorly understood. The facultative intracellular bacterium Legionella pneumophila is virulent in the stationary but not in the exponential growth phase, and therefore, an in-depth characterization of the pathogen's physiological states and ribosome profiles is crucial for understanding its virulence. Using bioinformatics, bacterial genetics, biochemical, and cell biological approaches, in this study, we reveal that the L. pneumophila ribosome hibernation factors LhpF, RaiA, RsfS, and HflX determine distinct ribosome subpopulations and are implicated in starvation survival and regrowth, as well as in host cell infection, intracellular replication, and phenotypic heterogeneity. Collectively, our data highlight the critical importance of ribosome hibernation for the physiology and virulence of L. pneumophila.

In Vibrio species, quorum sensing signaling culminates in the production of the master transcription factor SmcR that regulates group behavior genes in a density-dependent manner. Previously, we identified a small-molecule thiophenesulfonamide inhibitor called PTSP [3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole] that targets the SmcR family of proteins in multiple Vibrio species and blocks activity in vivo. Here, we used structure-function analyses to identify eight PTSP-interacting residues in the ligand-binding pocket that are required for PTSP inhibition of Vibrio vulnificus SmcR. Binding of PTSP to SmcR drives allosteric unfolding of the N-terminal DNA-binding domain, and, in this state, SmcR is specifically degraded by the ClpAP protease. This mechanism of PTSP inhibition was observed for all thiophenesulfonamide compounds tested against V. vulnificus as well as Vibrio parahaemolyticus and Vibrio campbellii. We show that Vibrio cells expressing degradation-resistant smcR alleles are impervious to changes in cell density state. These studies implicate ligand binding as a mediator of SmcR protein stability and function, which dictates the timing of quorum-sensing gene expression in three Vibrio pathogens.IMPORTANCESmcR family proteins were discovered in the 1990s as central regulators of quorum-sensing gene expression and later discovered to be conserved in all studied Vibrio species. SmcR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each SmcR-type protein has a predicted ligand-binding pocket. However, no native ligand has been identified for these proteins that control their function as regulators. Here, we used SmcR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation in vivo, providing the first demonstration of SmcR function connected to ligand binding for this historical protein family.

Mycobacterium tuberculosis uses several ESX type VII protein secretion systems for pathogenesis. M. tuberculosis ESX-5 is only partially characterized because it is essential for growth in standard lab culture conditions. To circumvent ESX-5 essentiality, we made an M. tuberculosis strain in which the central ESX-5 membrane component EccD₅ can be conditionally depleted. Here, we use this strain to demonstrate that M. tuberculosis requires the ESX-5 secretion system to grow using specific carbon sources in vitro, to grow in cultured macrophages, and to replicate and disseminate in aerosol-infected mice. M. tuberculosis requires ESX-5 to use glycerol or glucose as the sole carbon source. Use of glycerol and glucose also depends on the outer membrane protein PPE51. We show that M. tuberculosis requires ESX-5 activity for outer membrane export and surface exposure of PPE51. Expression of the outer membrane porin MspA enabled growth of ESX-5-deficient M. tuberculosis on glycerol, suggesting that the main function of ESX-5 in vitro is to export nutrient transporters to the outer membrane. Depletion of EccD₅ in acutely infected mice caused clearance of M. tuberculosis from lung tissues, demonstrating the critical importance of ESX-5 activity during infection. Our findings suggest that ESX-5 promotes M. tuberculosis pathogenesis by mediating export of outer membrane proteins that enable nutrient acquisition.IMPORTANCEMycobacterium tuberculosis ESX type VII secretion systems play important roles in pathogenesis, but the functions of ESX-5 are not well characterized because it is essential for growth in standard lab culture conditions. We used a strain that conditionally expresses a central membrane component of the ESX-5 secretion apparatus to determine how ESX-5 impacts growth in lab cultures and in a mouse infection model. We found that M. tuberculosis requires ESX-5 to grow using several carbon sources and to grow in the lungs of infected mice. Inhibiting production of the ESX-5 secretion system in mice also led to clearance of M. tuberculosis from lung tissues. Our results demonstrate that the M. tuberculosis ESX-5 system is a critical virulence factor and suggest that ESX-5 is a strong candidate for antitubercular drug development.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection and are associated with the development of cancer and multiple sclerosis. Unlike other viral families, gammaherpesviruses selectively target B cells to establish chronic infection. Specifically, gammaherpesvirus-driven differentiation of latently infected cells through the germinal center supports chronic infection and seeds viral lymphomagenesis. CD11c⁺ B cells are induced by most viral infections and are also observed in aged individuals and autoimmune diseases. Classically, CD11c⁺ B cells differentiate via an extrafollicular pathway that does not involve germinal center response, generating antibodies of beneficial (antiviral) or pathogenic (self-reactive) nature. While CD11c⁺ B cells are induced during B cell-tropic gammaherpesvirus infection, their role in chronic infection remains poorly defined. Here, we demonstrate that infection of the CD11c⁺ B cells, including those expressing germinal center markers, contributes to the overall latent gammaherpesvirus reservoir during natural infection. Both T-bet⁺ and T-bet^neg CD11c⁺ B cell subsets expanded and underwent germinal center differentiation during chronic gammaherpesvirus infection. Furthermore, B cell-intrinsic T-bet expression attenuated the long-term latent viral reservoir, gammaherpesvirus-driven germinal center responses, and differentiation of self-reactive B cells. In summary, our study for the first time defines CD11c⁺ splenic B cells as a reservoir of latent gammaherpesvirus during mucosal chronic infection and reveals an important role of T-bet⁺ B cells in controlling long-term infection and gammaherpesvirus-driven pathogenic host processes.IMPORTANCEGammaherpesviruses are ubiquitous pathogens that are associated with cancer and multiple sclerosis. These viruses selectively infect B cells and drive their differentiation through the germinal center response to establish chronic infection. Here, we demonstrate that gammaherpesvirus infection drives expansion and germinal center-based differentiation of CD11c⁺ B cells that host the latent viral reservoir. We also show that B-cell-intrinsic T-bet expression is important for control of long-term gammaherpesvirus infection and pathogenesis.

Excessive binding of antibodies to the bacterial cell surface can paradoxically increase resistance of some Gram-negative pathogens to complement-mediated killing (CMK). We examined the CMK of 336 Neisseria gonorrhoeae isolates from 283 participants recruited to a clinical trial. Serum bactericidal assays revealed 3% (9/283) of the autologous participant sera blocked CMK. Gonococci isolated from these participants were resistant to the autologous host serum, but sensitive to pooled healthy control sera (HCS) and protected by autologous host serum in a 1:1 mixture with HCS. Analysis of clinical metadata showed that there was a significantly higher proportion of blocking sera found in participants with urethral infection and from men within the transmission network of men who have sex with women, when compared to the whole cohort. Following antibody purification from participants with blocking sera (5/9), total IgG protected autologous isolates from HCS-mediated killing. A closer examination of IgG subclasses using whole gonococcal cell ELISAs revealed a significant correlation between increased IgG2 binding and decreased IgG3 binding to the cell surface of isolates that were resistant to CMK. This indicates that IgG2 prevents bactericidal IgG3 from initiating CMK, with an increased IgG2:IgG3 ratio blocking CMK of gonococci. We therefore reveal a previously unrecognized mechanism by which blocking antibodies prevent CMK of N. gonorrhoeae.

Importance: The antigenic variation of Neisseria gonorrhoeae and a limited mechanistic understanding of immune responses to this bacterium have presented multiple challenges to generating a protective vaccine. Here, we use a collection of N. gonorrhoeae clinical isolates (n = 336) for a robust analysis of the host immune response to infection. We reveal a mechanism for serum resistance in which some isolates of N. gonorrhoeae drive the production of inhibitory IgG2 antibodies, which block the activity of IgG3 bactericidal antibodies. Importantly, an increased ratio of IgG2:IgG3 bound to the bacterium promotes serum resistance. Recently, there has been increased interest in developing a vaccine against N. gonorrhoeae given the observation that the licensed outer membrane vesicle-based vaccine against Neisseria meningitidis (MeNZB) generated some cross-protection against N. gonorrhoeae. Thus, the mechanism described here should guide the development of a vaccine that simultaneously prevents serum resistance and promotes serum killing of the gonococcus.

Degenerative genome evolution is widely found among obligatory bacterial mutualists, as observed in plant-sucking hemipteran insects whose symbiont genomes are highly reduced and specialized for provisioning of essential amino acids. Originally, such symbionts must have been derived from environmental free-living bacteria. It is elusive, however, what evolutionary changes are involved in the early stages of such elaborate mutualistic associations. Here, we addressed this evolutionary question using the experimental symbiotic system consisting of the stinkbug Plautia stali and the model bacterium Escherichia coli. In E. coli, metJ encodes a repressor of the methionine synthesis pathway, and its disruption upregulates production of the essential amino acid methionine. We found that, when metJ-disrupted E. coli was inoculated to P. stali, the insects exhibited significantly elevated hemolymphal methionine levels and improved adult emergence rates, demonstrating that the single-gene mutation makes E. coli mutualistic to P. stali. In comparison with mutualistic E. coli single-gene mutants that upregulate another essential amino acid tryptophan, the phenotypic effects on P. stali were somewhat different: the adult emergence rate was improved by both the methionine-overproducing and tryptophan-overproducing E. coli mutants, whereas the adult body color was improved by the tryptophan-overproducing E. coli mutant only. When we generated a double mutant E. coli ΔmetJΔtnaA and inoculated it to P. stali, the adult emergence rate was not improved but rather attenuated, uncovering non-additive fitness consequences of these single-gene mutations. These results provide insights into what genetic changes may have facilitated the early evolution of the insect-microbe mutualism.IMPORTANCEWhat is the evolutionary origin of elaborate bacterial mutualists entailing drastic genome reduction, specialized metabolism, and uncultivability? This question is important but challenging to address, because the evolution of such symbiotic associations occurred in the past and cannot be observed directly. However, the recent development of an experimental symbiotic system consisting of the stinkbug Plautia stali as host and the model bacterium Escherichia coli as symbiont has opened an avenue to empirically investigate the evolution of host-microbe mutualism. We demonstrated that, strikingly, single-gene mutations of E. coli that upregulate the production of methionine and tryptophan make the non-symbiotic bacterium mutualistic to P. stali, plausibly via provisioning of the essential amino acids that complement the nutritional requirements of the plant-sucking insect host. Our finding provides insight into what genetic changes of the symbiont side can be involved in the early evolution of the host-microbe mutualism.

Extracellular vesicles (EVs) play crucial roles in fungal communication and host immune modulation, representing potential therapeutic targets for fungal infections. This study investigated the role of fungal EVs in both intra- and interspecies communication, focusing on their effects on virulence and immune responses. Co-incubation experiments were performed using EVs derived from Candida albicans and Candidozyma auris to assess interactions with C. albicans planktonic cells and biofilms, as well as Cryptococcus neoformans and Cryptococcus gattii EVs interacting with C. neoformans cultures. EVs were observed associating with recipient cell surfaces, suggesting subsequent internalization. Functional assays revealed that EV exposure led to increased expression of CAP59, LAC1, URE1, and ERG11 genes, correlating with reduced antifungal susceptibility in both planktonic and biofilm forms. Additionally, EVs facilitated cross-species communication, enhancing biofilm adhesion and dispersion, which emphasizes their role in phenotypic modulation. Macrophages stimulated with fungal EVs exhibited receptor-specific gene expression changes (dependent on the EVs' origin, including variation among species of the same genus), along with a pro-inflammatory phenotype marked by increased iNOS expression, enhanced TBK1/STAT1 production, and elevated levels of IL-1β, IL-6, and IL-8. Collectively, these findings underscore a critical role for fungal EVs in interspecies communication, biofilm regulation, and immune modulation, offering valuable insights into fungal pathogenicity mechanisms.IMPORTANCECurrently, no vaccines exist to prevent fungal infections, underscoring the need for new therapies. As fungal diseases increase globally, understanding fungal biology is essential to identifying treatment targets. Fungi use extracellular vesicles (EVs) to communicate and evade immune responses. EVs mediate cell-cell communication, transporting proteins, polysaccharides, lipids, and nucleic acids, serving as "messages" exchanged within a fungal network. Understanding how these vesicles facilitate communication not only within a single species but also across different fungal species can shed light on their contribution to infection persistence and cross-species adaptability. Moreover, EVs may have a broader role in inter-kingdom signaling, influencing how fungi interact with host immune cells. The impact of fungal EVs on human innate immune responses remains a largely underexplored area, with significant gaps in our knowledge. This study aims to examine how fungal EVs affect immune responses and whether their signaling varies across species, potentially revealing new therapeutic targets.