PLOS Pathogens

Like most herpesviruses, KSHV encodes multiple microRNAs (miRNAs). Collectively, they comprise an important mechanism through which the virus maintains latency and persists in cells. At the same time, individual miRNAs can also play distinct, nonredundant roles. Past experiments with single miRNA knockout viruses showed that miR-K12-9, in particular, filled a unique niche. Endothelial cells latently infected with the miR-K12-9 knockout grew to be many times larger than WT-infected cells and proliferated at a significantly slower rate. Their ability to migrate was slowed as well. RNA-seq identified nearly 8,500 differentially expressed genes between miR-K12-9 knockout- and WT-infected cells. To further study miR-K12-9, we generated Telomerase-Immortalized Microvascular Endothelial (TIME) cells expressing either miR-K12-9 or a control miRNA from a lentivirus. Unexpectedly, after approximately one month in culture, unmistakable morphological changes began to occur in two of the three miR-K12-9-expressing cell lines. These smaller, more rounded cells proliferated rapidly and swiftly took over the two cultures. Given this result, we proceeded to characterize all the lentivirus-transduced cell lines in various assays focused on oncogenesis. When looking at colony formation in soft agar, only those two miR-K12-9-expressing cell lines produced colonies, indicating a loss of contact inhibition. NOD/SCID mice injected with the two cell miR-K12-9-expressing cell lines developed tumors while those receiving other cell lines did not. To confirm reproducibility of these results, we transduced both TIME and primary endothelial cells (HUVECs) with the miR-K12-9 and control lentiviruses. Once again, approximately half of the cell lines expressing miR-K12-9 showed hallmark phenotypes of transformation. We are currently characterizing the miR-K12-9 targetome in the transduced cell lines and mouse tumors using bulk and single-cell RNA-seq. This should yield insights into the underlying mechanism and required cofactors of miR-K12-9-induced transformation. To our knowledge, this is the first description of transformation of endothelial cells by a viral miRNA.

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

Persistence - characterized by the transient ability of subpopulations of drug-susceptible parasites to survive exposure to drug - is a major driver of treatment failure and clinical relapses in leishmaniasis. Persisters are characterized by non-dividing or slow-growing state and increased drug tolerance. However, the molecular mechanisms governing formation of persisters remain poorly understood in Leishmania parasites. Here, we developed a model to explore persistence in Leishmania mexicana. Viable promastigote persister-like subpopulations were enriched using Ficoll density gradient centrifugation following lethal exposure to antimonial drugs that killed 80% of parasites. The surviving parasites exhibited delayed growth in drug-free medium that is characteristic for persisters, and significantly higher tolerance to drug upon rechallenge. Transcriptomic profiling across acute stress, drug-free recovery, and rechallenge phases revealed a global remodeling in persisters under all tested conditions. Induction phase in Leishmania persisters was characterized by downregulation of several biological processes and a robust upregulation of nucleolar pathways, supporting epitranscriptomic changes during formation of persisters. Upon drug removal, this profile rapidly reverted, initiating ribosomal biogenesis to exit latency and resume cellular proliferation. Resuscitation phase exhibited activated protein synthesis and upregulation in many biological processes associated with metabolic and mitochondrial functions. Furthermore, comparative analysis of drug responses in rechallenged drug-tolerant persisters and parental parasites exposed to the drug for the first time, revealed that persisters exhibit distinct drug response profiles compared to parental parasites by rapidly implementing a highly conserved, coordinated survival reprogramming, where 316 genes were uniquely downregulated, and 241 genes were upregulated. The distinct features of the drug response in rechallenged persister cells were characterized by the downregulation of mitochondrial function and protein synthesis machinery to induce a dormant, idling state, and the upregulation of drug-response and stress-tolerance genes to survive immediate toxicity. In contrast, parental parasites displayed a broad and disorganized drug response. Additionally, rechallenged persisters exhibited a distinct transcriptomic memory that transiently phenocopies stable genetic resistance. This pre-adapted state is characterized by the targeted upregulation of epigenetic modulators, heavy metal transporters, and catabolic enzymes to maintain viability. These findings demonstrate that drug persistence in Leishmania is not merely a metabolic collapse, but rather a sophisticated survival strategy involving active transcriptome remodeling, downregulation of translation and epigenetic adaptations. This transient state constitutes an initial evolutionary step toward permanent drug resistance and highlights new molecular vulnerabilities for therapeutic interventions aimed at preventing clinical relapse. Author summaryThe efficacy of leishmaniasis chemotherapy is often fails because Leishmania parasites survive the drugs by entering a dormant state, causing relapses in patients. In this study, however, we discovered that these parasites are not simply quiescent. On the contrary, they orchestrate a highly active survival response: they shut down energy-consuming functions while keeping their cellular alert mechanisms active. Remarkably, upon re-exposure to antileishmanial drugs, the parasites recall previous stress events and transiently activate defensive pathways characteristic of stable drug resistance. Understanding how Leishmania employs this temporary adaptive strategy provides critical targets for developing therapies designed to eradicate the infection and avert relapses.

The matrix protein VP40 of orthoebolaviruses coordinates virion release and downregulates viral RNA synthesis through distinct oligomeric states, including dimers, octamers, and filamentous assemblies. To dissect the contributions of two oligomeric interface residues, L117 and W95, in the Sudan virus (SUDV) VP40 (sVP40), we created variants carrying alanine substitutions and assessed their structural and functional properties. sVP40 L117A failed to form dimers and was predominantly monomeric showing increased structural flexibility, reduced thermal stability together with loss of plasma membrane transport, budding activity, and the ability to regulate viral RNA synthesis. VP40 W95A preserved dimerization but also exhibited increased structural flexibility and reduced thermal stability. Functionally, sVP40 W95A more strongly inhibited viral RNA synthesis and markedly enhanced budding. However, in a transcription- and replication-competent virus-like particle (trVLP) assay, trVLPs produced with sVP40 W95A induced substantially reduced reporter activity in target cells, indicating impaired particle infectivity or functionality and suggesting possible defects in minigenome packaging, entry, or early post-entry steps. These results demonstrate that mutations at key oligomerization interfaces exert distinct structural and functional effects and highlight the requirement for precise oligomerization in coordinating sVP40s dual roles in genome regulation and virion release. By defining the contributions of L117 and W95, this study advances mechanistic understanding of sVP40 function and identifies processes that may serve as targets for antiviral intervention. ImportanceSudan virus (SUDV) causes regular outbreaks in Sub-Sahara Africa with unusually high lethality rates. However, in contrast to the more often occurring Zaire ebolavirus (EBOV), no monoclonal antibodies or vaccines are available and SUDV is generally understudied. The matrix protein VP40 is responsible for the downregulation of viral genome replication and transcription as well as budding. Here, we present structural and functional characterization of the SUDV VP40 interface residues L117 and W95 and show that while both amino acids are crucial for VP40s structural integrity, their functional effects are dramatically different ranging from complete abolishment to improving regulatory and budding activities.

Entamoeba histolytica is a protozoan parasite that can cause severe liver disease known as amoebic liver abscess. However, only a subset of infected individuals develops invasive disease, indicating that host-parasite interactions are critical determinants of disease outcome. In this study, we investigated the clone-specific modulation of hepatic immune responses using non-pathogenic A1np and pathogenic B2p E. histolytica clones. Time-resolved transcriptome analyses (6, 12, 24 hours post-infection) in a murine model revealed distinct immune trajectories. Both clones activated innate immune pathways early after infection, but their responses differed markedly in magnitude and composition. A1np infection induced a rapid and controlled inflammatory response associated with antimicrobial activity and resolution-promoting signalling. In contrast, B2p infection triggered a stronger and more complex immune response characterised by pronounced cytokine and chemokine expression, activation of stress and redox pathways, and tissue remodelling processes. The B2p induced response exhibited features of excessive immune activation, accompanied by the upregulation of counter-regulation genes such as Ackr2. These findings indicate that liver pathology is not solely determined by parasite presence, but rather may also be influenced by the nature and regulation of the host immune response. Overall, the observed differences between A1np and B2p infections suggest that parasite-specific properties shape hepatic immune activation and may influence disease progression. Author summaryAlthough infection with the parasite Entamoeba histolytica can lead to severe liver disease, most infected individuals remain asymptomatic. This suggests that the outcome of the disease is not determined solely by the parasite, but also by how the host responds to the infection. In this study, we used a mouse model to compare how the liver reacts to infection with two E. histolytica clones that differ in their ability to cause amoebic liver abscesses. Using this model and time-resolved transcriptome analysis, we found that both clones trigger an early immune response; however, the nature of this response differs markedly. The non-pathogenic clone induced a rapid and controlled reaction associated with antimicrobial defence and tissue protection. In contrast, the pathogenic clone provoked a stronger and more prolonged inflammatory response accompanied by cellular stress and tissue remodelling processes. Notably, this heightened response also activated regulatory mechanisms that attempted to limit excessive inflammation. Our findings demonstrate that differences in disease severity are linked to the activation and regulation of the host immune system, rather than simply to the presence of the parasite.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus burden at 3 h post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support early infection by B. abortus and B. ovis without impairing general phagocytosis. Gene set enrichment analysis further revealed positive regulators of mTORC1 signaling as key host factors; this result was validated through targeted disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Thus, the AKT-Ragulator-mTORC1 signaling axis contributes to the establishment of a permissive intracellular niche during early Brucella infection. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that support early Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.

Recent studies demonstrated that the structural maintenance of chromosomes complex 5/6 (SMC5/6) acts as an important antiviral restriction factor that silences episomal DNA. Consequently, viruses have evolved strategies to antagonize this defence system. Interestingly, it was shown that SMC5/6-mediated silencing of extrachromosomal DNA depends on its recruitment to PML nuclear bodies which is mediated by a protein termed SMC5/6 localization factor 2 (SLF2). Since human cytomegalovirus (HCMV) dissociates PML nuclear bodies (PML-NBs) during the first hours of infection, we asked for the fate of SMC5/6 components during the HCMV replicative cycle. We first investigated the expression levels of SMC5/6 core components at the onset of HCMV infection and found that they were not downregulated by HCMV. Instead, we observed a distinct decrease of SLF2 protein levels which correlated with a complete dispersal of the SMC5/6 complex from PML-NBs. This was also observed after infection with UV inactivated HCMV and could not be blocked by cycloheximide treatment suggesting the involvement of an imported structural component of the virion. While we could exclude a role of the HCMV tegument protein pp71 (UL82), a targeted screen identified the tegument protein UL35 as being required and sufficient for proteasomal degradation of SLF2. In line with these results, SLF2 levels remained stable after infection with a recombinant HCMV harbouring a stop codon within the UL35 gene region and this correlated with a defect in the onset of HCMV immediate early gene expression. Furthermore, we observed an enhanced recruitment of SMC5/6 to PML-NBs and an overall increase in the association of parental viral genomes with SMC5/6 containing PML-NBs in the absence of UL35. Congruently, siRNA mediated depletion of SLF2 resulted in a reversal of this phenotype. This strongly suggests that UL35 serves as a novel viral SLF2 antagonist to prevent the initial silencing of incoming viral genomes by SMC5/6. Author summaryHuman cytomegalovirus (HCMV) is an important human pathogen which establishes a life-long persistent infection. Previous research revealed that host intrinsic defense mechanisms can silence HCMV gene expression and thereby foster latency. However, during lytic infection, viral effector proteins like the structural protein pp71 (UL82) and the viral immediate-early protein IE1 act as antagonists of this defense. Here, we report on the identification of a novel antagonist of host-mediated viral gene silencing which targets the SMC5/6 complex. SMC5/6 belongs to a highly conserved family of protein complexes that act as loop extrusion machines to facilitate the folding of chromatin. While SMC5/6 core factors were not attacked by HCMV, we found that the SMC5/6 associated protein SLF2 was degraded by the viral tegument protein UL35, that is imported into infected cells. This abrogates the accumulation of SMC5/6 at PML nuclear bodies which compromises the entrapment of viral genomes in these subnuclear structures. Since SLF2 is also important for genome stability and pathogenic variants of SLF2 cause the neurodevelopmental disease Atelis syndrome, we speculate that depletion of this factor via a viral tegument protein may also contribute to DNA damage induced during congenital HCMV infection.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome by disrupting the intestinal brush border. During infection, EHEC injects the transmembrane virulence factor Tir into enterocytes; upon insertion into the apical membrane, this factor mediates bacterial attachment and drives formation of actin-rich pedestals needed for colonization. How Tir is inserted into the host plasma membrane remains unclear. Here, we investigated the role of brush border resident IRTKS, a Tir- and membrane-binding protein, in this process. Using multiple IRTKS gain- and loss-of-function models, we analyzed pedestal organization and component localization. Whereas canonical models position IRTKS downstream of Tir as a scaffolding link to F-actin, we found that perturbing IRTKS disrupted the distribution and abundance of Tir. Moreover, ectopic IRTKS expression enhanced Tir membrane insertion in the absence of other virulence factors. We conclude that IRTKS functions early in pedestal formation to promote Tir accumulation in the plasma membrane and in turn, facilitate bacterial attachment. SUMMARYEHEC attaches to intestinal epithelial cells using injected virulence factor Tir, which forms actin pedestals and promotes bacterial colonization. We found that host protein IRTKS promotes Tir accumulation in the plasma membrane, to facilitate intimate bacterial attachment and pedestal formation.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.

Immediate-early protein IE1 is an essential protein of baculoviruses that is involved in transcription and replication. In the present study, we provide several lines of evidence for how ancient IE1 evolved into its current version. Using progressive truncations and site-directed mutations coupled with fluorescence microscopy, surprisingly, we showed that the previously identified nuclear localization sequence (NLS), basic domain II, was essential for sequence non-specific DNA binding, but not required for IE1 nuclear import, and demonstrated that, BmNPV IE1 (BmIE1) possesses not only a unique bipartite NLS that contains a monopartite NLS but also a cryptic non-canonical NLS that is correlated with sequence non-specific DNA binding. The non-canonical NLS alone was sufficient to launch infection. We also found that N-terminally truncated IE1 can enter the nucleus through its sequence non-specific binding ability. Remarkably, the monopartite NLS, when fused to the N-terminus of EGFP, can form a novel NLS that is also functional in a mammalian cell line. Moreover, residues 58 to 151 of BmIE1 were shown to be dispensable, and residue 152 was found to be critical for launching a productive infection. To gain insight into how ancient IE1 acquired its multi-functionality, we reorganized the N-terminal 23 aa and 132 aa of BmIE1 with EGFP in a variety of manners and found that both fragments are separable and transferable. Notably, we observed that the nuclear levels of BmIE1 should reach certain thresholds to initiate infection. Additionally, we found that BmNPV could launch infection more efficiently in a BmN cell line over another BmN cell line by increasing transcription levels of immediate early genes. Collectively, these findings suggest a hypothesis where ancient IE1 might have evolved the two additional NLSs and acquired the N-terminal 132 aa through gene fusion so as to reach infection-initiating thresholds at a faster pace. Author SummaryBasic domain II was previously shown to be the NLS of AcMNPV IE1 protein. In the present study, we demonstrated that it is essential for sequence non-specific DNA binding, but not required for IE1 nuclear import, and found that BmNPV IE1 (BmIE1) possesses not only a unique bipartite NLS that contains a monopartite NLS but also a cryptic non-canonical NLS that is correlated with sequence non-specific DNA binding. We also found that N-terminally truncated BmIE1 can enter the nucleus through its sequence non-specific binding ability. Remarkably, the monopartite NLS, when fused to the N-terminus of EGFP, can form a novel NLS that is also functional in a mammalian cell line. Moreover, the domains of BmIE1 was shown to be separable and transferable. We also demonstrated that higher expression of functionally impaired BmIE1 achieved by higher MOIs or higher transfection efficiency can partially complement its compromised functions. Consistently, we found that BmNPV could launch infection more efficiently in a BmN cell line over another BmN cell line by increasing transcription levels of immediate early genes.

Klebsiella pneumoniae is a leading cause of pneumonia and bacteremia and is especially dangerous in healthcare settings. Despite massive clinical significance, the mechanisms used by macrophages to kill K. pneumoniae are not well defined. Macrophages are critical for controlling K. pneumoniae as mice lacking monocyte-derived or alveolar macrophages have higher bacterial tissue burdens and mortality. Two prominent mechanisms used by macrophages to kill bacteria are the production of reactive oxygen species (ROS) via the NADPH oxidase NOX2 and reactive nitrogen species (RNS) via the inducible nitric oxide synthase iNOS. Previously, we found that K. pneumoniae uses similar genetic factors to survive during bacteremia and within macrophages. The ability of these factors to enhance intracellular fitness was significantly correlated with resistance against RNS, not ROS. Here, we aimed to define whether macrophage ROS and RNS contribute to intracellular K. pneumoniae clearance. Using wild-type, Cybb-/-, and Nos2-/- cells, we measured K. pneumoniae survival within macrophages lacking such defenses. NOX2 was dispensable for K. pneumoniae clearance, and ROS was undetectable in K. pneumoniae-infected macrophages. We confirmed that ROS was undetectable within alveolar-like macrophages, indicating a conserved ROS evasion phenotype across macrophage subsets. Instead, iNOS significantly contributed to macrophage clearance of K. pneumoniae and enhanced cytokine production. iNOS likely enhances K. pneumoniae clearance through coordination of immunity and RNS. Activation of pathways upstream of iNOS may be the most relevant to supporting effective macrophage control of K. pneumoniae. This study defines unexpected differential roles for ROS and RNS in macrophage clearance of K. pneumoniae.

How host functions affect resistance to antiviral drugs is poorly understood. Ganciclovir, a chain-terminating nucleoside analog, is a first-line therapy against human cytomegalovirus, a widespread herpesvirus that causes life-threatening disease in immunocompromised individuals and newborns. Ganciclovir resistance, which is caused by mutations that affect the viral kinase, UL97 and/or the viral polymerase, UL54, can cause treatment failures. Among these mutations, those reducing the exonuclease activity of the viral DNA polymerase permit ganciclovir incorporation without chain termination. However, the fate of DNA strands containing the incorporated nucleotide analog is unknown. We show here that template DNA containing ganciclovir fails to support DNA synthesis of the complementary strand by exonuclease-mutant polymerase. Moreover, while DNA synthesis and ganciclovir incorporation are limited in drug-treated fibroblasts infected by virus with wild-type polymerase, an exonuclease-resistant mutant virus can better synthesize full-length genomes and incorporate substantially more ganciclovir into DNA. Notably, ganciclovir is lost from DNA when drug is removed, suggesting that ganciclovir-containing templates are repaired. We identify the host nucleotide excision repair component, XPA, and the repair enzyme, polymerase kappa, as each being necessary for mutant virus ganciclovir resistance and polymerase kappa as being required for the mutants cidofovir resistance, demonstrating a role for host DNA repair machinery in a mechanism of antiviral resistance. We propose a model for this mechanism, which has relevance for at least one other antiviral drug and likely other nucleoside analog therapeutics, and highlights the participation of host DNA repair machinery during human cytomegalovirus DNA replication. IMPORTANCENucleoside analogues such as ganciclovir, which is a leading drug for preventing and treating human cytomegalovirus, are a critical defense against viral diseases, but antiviral resistance often results in treatment failures. This study reveals a critical role for host DNA repair in a mechanism of resistance to ganciclovir, and identifies at least one specific repair pathway that permits viral DNA synthesis in the presence of ganciclovir, defining a mechanism by which cellular DNA repair pathways conspire to enable antiviral drug resistance. This mechanism is relevant to at least one other antiviral drug and may apply to other antiviral and anticancer agents. The study also showcases the participation of host DNA repair machinery during human cytomegalovirus DNA synthesis.

Many intracellular pathogens have evolved to evade immune responses and establish a secure niche inside host cells. One such stealth pathogen is the obligate intracellular bacterium Coxiella burnetii, the causative agent of Q-fever. Coxiella translocates an array of bacterial proteins ( effectors) into the host cell through a type IVB secretion system (T4BSS) that mediates suppression of pathogen sensing and innate immunity. Yet, at a systemic level, immunocompetent hosts often restrict pathogens through Th1-mediated and cell-autonomous immunity through the expression of immune-inducible genes. However, the expression and regulation of chemokines, particularly, the CXC-ligands (CXCL9,-10,-11) that are considered biomarkers of Q-fever, is poorly understood. We observed minimal to no CXCL10 transcript levels during Coxiella infection. However, Coxiella-infected cells robustly augmented IFN{gamma}-activated expression of CXCL10 in both phagocytic and non-phagocytic cells, and this process was dependent on viability and T4BSS in epithelial cells. This phenomenon extends to other highly pro-inflammatory cytokines and other pathogens including Salmonella, Mycobacteria (H37Ra) and Toxoplasma. Synergistic increase in CXCL10 expression in Coxiella-infected, IFN{gamma}-activated cells requires ISRE and NF-{kappa}B transcriptional elements in the promoter, and the transcription factors STAT1, STAT3 and IRF9. Inhibition of STAT3 by small molecule inhibitors potently decreased the excess promoter activity of CXCL10. In addition, treatment of Coxiella-infected cells with IFN{gamma} is associated with decreased expression of SOCS1, a negative regulator of the IFN{gamma} signaling axis and relatively higher detection of extracellular bacteria. Altogether, these data demonstrate that intracellular pathogens including those conventionally considered to be "immunologically silent", robustly synergize with IFN{gamma} signaling, with STAT3 activation emerging to be a nodal point for promoting both persistent infection as well as synergism in the expression of immune genes. Author summaryAcute host immune response is often associated with production of soluble messenger molecules called cytokines/chemokines which direct the migration, recruitment and activation of leukocytes and serve as biomarkers in infectious and inflammatory diseases. The regulation of expression of these molecules and their influence on the infection process is not well-understood. In particular, interferon-gamma (IFN{gamma}), a potent pro-inflammatory cytokine produced by activated T and NK cells, activates signaling pathways involved in host defense and inflammation in macrophages and other cell types. We observed that infection with many intracellular bacterial/parasitic pathogens that employ sophisticated immune evasion strategies, synergize with IFN{gamma} signaling and significantly amplify the levels of pro-inflammatory mediators implicating the origin of adverse immune pathologies. We investigated the mechanistic basis of this seemingly counter-intuitive phenomenon, underlying host and bacterial factors involved in distinct cell types, and identified the small molecule-targetable-transcription factor STAT3 as a host determinant in promoting excess cytokine synthesis.

Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H-ATPase (V-H+-ATPase), Ca{superscript 2}-transporting P-type ATPase (Ca2+-ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites. Author summaryProtozoan parasites such as Leishmania and Trypanosoma cause serious diseases affecting millions of people worldwide. To better understand how these parasites survive environmental changes during transmission between hosts, we studied a specialised organelle called the acidocalcisome, which stores polyphosphates and helps regulate stress responses. In this work, we used the non-pathogenic Leishmania tarentolae as a safe and cost-effective model that shares key cellular features with disease-causing species. Using a combination of CRISPR-Cas9 genome editing, proximity-based labelling (BioID), confocal microscopy, pulldown assays and AlphaFold3 structure prediction, we investigated the vacuolar transporter chaperone (VTC) complex, which synthesises and transports polyphosphate into the acidocalcisome lumen. Proximity proteomics identified several known proteins located near the VTC complex, and importantly, led us to discover three novel proteins that interact with it. These findings open new directions for exploring the organisation and regulation of the VTC complex in protozoan parasites. By revealing novel protein interactions, our study contributes to a deeper understanding of parasite biology and may help identify therapeutic targets for treating neglected tropical diseases.

Coxiella burnetii is the only member of the order Legionellales known to primarily infect vertebrates. The Q fever pathogen is also unusual in that it replicates within an acidified phagolysosome-like vacuole. The evolutionary origins of the virulence determinants underlying this lifestyle remain unclear. More broadly, little is known about how virulence-related traits arise in specialized intracellular lineages, where access to foreign-origin DNA may be more episodic. To address this question, we used Legionellales-wide comparative phylogenomics to reconstruct the gain and loss of traits affecting host interaction, immune evasion, intracellular survival, and metabolism. We found that many virulence-associated traits in C. burnetii predate the modern pathogen and were assembled stepwise in ancestors that likely occupied niches distinct from the acidified vacuolar niche of modern C. burnetii. The common ancestor shared with soft-tick Coxiella endosymbionts likely encoded most C. burnetii type IVB secretion system effectors, indicating that much of the host-manipulation repertoire in C. burnetii was already present before the emergence of the modern pathogen. Distinctive lipopolysaccharide features associated with immune evasion also appear to have accumulated progressively within the Coxiella lineage, including genes implicated in synthesis of virenose, a unique O-antigen sugar critical for C. burnetii virulence. Traits likely to support replication in the acidic Coxiella-containing vacuole likewise accumulated gradually, with generalized stress-tolerance functions predating acquisition of an Mrp cation/proton antiporter that may have further supported pH homeostasis. Additional changes in sugar transport and catabolism, glycolytic control, and respiratory metabolism likely enhanced metabolic flexibility and access to diverse substrates in this nutrient-rich niche. Together, these findings support a model in which vertebrate pathogenicity in C. burnetii emerged through stepwise remodeling of an ancestral host-associated lineage and provide a framework for understanding how virulence-related traits evolve in specialized intracellular pathogens. AUTHOR SUMMARYCoxiella burnetii is the bacterium that causes Q fever, a disease that can spread from animals to humans. Unlike its close relatives, C. burnetii primarily infects vertebrates and grows inside an acidic compartment within host cells. New bacterial pathogens often evolve by gaining genes from other bacteria, but how virulence evolves in lineages that grow only inside host cells, where opportunities to gain new genes may be infrequent, remains unclear. We wanted to understand how C. burnetii evolved the traits needed for its distinctive intracellular lifestyle. By comparing its genome to those of related bacteria across the order Legionellales, we found that features involved in host manipulation, immune evasion, acid tolerance, and nutrient use appeared at different times in its ancestry rather than being acquired all at once by the modern pathogen. Our findings suggest that specialized intracellular pathogens can emerge through gradual changes in ancestral host-associated lineages, including gene acquisition, gene loss, retention of older traits, and repurposing of existing functions.

Despite advances in knowledge and medicine, hepatitis C virus (HCV) infection remains a global challenge. The viral life cycle heavily depends on lipid metabolism; therefore, HCV infection is associated with profound changes in host lipid homeostasis. The transcription factor nuclear factor erythroid 2 related factor-1 (Nrf1) is one of the regulators maintaining this homeostasis. Nrf1 exists in multiple proteoforms that differ in their capacity to serve as cholesterol sensor, activator or inhibitor of gene expression. We have previously identified that the amount of full-length Nrf1 protein in HCV-replicating cells is significantly reduced. Here, we investigate whether HCV affects the formation of the different proteoforms and their functionality using Western blot, qPCR, CLSM and FRET acceptor-photobleaching methods. We report that HCV infection does not alter the onset of Nrf1 proteoforms generated through proteasomal cleavage of the protein. However, the amount of different Nrf1 proteoforms is significantly reduced in HCV-positive cells due to enhanced Nrf1 turnover. Furthermore, the Nrf1 proteoforms with transcriptional activator functions are prevented from translocation into the nucleus. Reduced Nrf1 activity contributes to elevated cholesterol levels and favors lipid droplets formation, which serve as a central platform for viral morphogenesis. Conversely, rescue of Nrf1 activity in HCV-replicating cells is associated with decreased intracellular cholesterol levels, reduced number of lipid droplets and impaired viral release, which is reflected by intracellular accumulation of the core protein and intact viral particles. Taken together, our results characterize the so far not investigated complex interplay between HCV and Nrf1. HCV-mediated inhibition of Nrf1 functionality leads to intracellular cholesterol accumulation, resulting in enhanced lipid droplet formation that supports the HCV life cycle and contributes to HCV-associated pathogenesis. Author SummaryThe lack of a vaccine and limited access to effective drugs (pan-genotypic direct-acting antivirals) for curing hepatitis C virus (HCV) infection means that HCV remains an ongoing and urgent challenge worldwide. In light of this, a deeper understanding of the virus-host interaction is required. In this study, we investigate the interplay between HCV and lipid metabolism, focusing on the uncharacterized role of the cholesterol sensor and transcription factor Nrf1 in this interaction. We observe the inhibition of Nrf1 activity in HCV-replicating cells, which leads to enhanced intracellular cholesterol accumulation and lipid droplet formation, resulting in microenvironment favorable for viral morphogenesis. We reveal the underlying mechanisms and describe their relevance to the viral life cycle and virus-associated pathogenesis.

Type III secretion systems (T3SS) are common virulence factors that facilitate the injection of anti-eukaryotic effector toxins that damage and kill target host cells. The sequence and function of the structural and regulatory proteins of these systems are conserved across Gram-negative pathogens, including Pseudomonas, Yersinia, and Salmonella. However, the identity and function of effector proteins are not conserved and are unknown in several relevant human and animal pathogens. Here, we used comparative genomics to identify and characterize the effectors encoded by Vibrio campbellii BB120, a crustacean and fish pathogen. We showed that most sequenced Vibrio strains belonging to the Harveyi clade, including V. campbellii, encode a full set of structural and regulatory genes corresponding to the T3SS1 of V. parahaemolyticus; the exception is V. natriegens. Transcriptomic and proteomic analyses identified four V. campbellii effectors that are secreted by the T3SS. Among these, two effectors are encoded outside the T3SS island, and all effector genes were co-regulated by both the master T3SS regulator ExsA and by the master quorum sensing regulator LuxR. Three effectors - VopS, CopA, and VIBHAR_06684 - exhibited toxic activity in yeast cells or bone marrow-derived macrophages, and the toxicity phenotypes were dependent on a functional T3SS. VopS and CopA are conserved among the queried species of the Harveyi clade. VIBHAR_06684 or VIBHAR_05674 did not show conservation among the queried species. These findings demonstrate that T3SSs in bacteria from the same clade have conserved structural secretion apparatuses but exhibit variance in effector repertoires. We postulate that the functions of effectors differ between species to impart roles in host specificity.

The apicoplast of malaria parasites retains a reduced genome encoding a small set of genes with unknown functions. Among these genes is a putative ClpM chaperone, which unlike other apicoplast Clp-family members is not nuclear but plastid-encoded. In this study, we used ClpM as a model case to investigate evolutionary and molecular basis for plastid genome retention. Phylogenetic analyses across plastid-containing eukaryotes revealed that ClpM orthologues are broadly conserved and consistently plastid-encoded in all organisms with a red alga-derived plastid, irrespective of parasitism, photosynthesis or physiology. This broad phenomenon suggested gene-specific evolutionary constraints that were subsequently tested experimentally. To test whether clpM can be functionally expressed from the nucleus, we generated transgenic parasites carrying a nuclear ClpM copy fused to an apicoplast-targeting transit peptide. Unexpectedly, standard transgenesis resulted in transcriptional silencing, and we therefore forced transcription using integration into an endogenous essential locus. This led to robust clpM mRNA, however no detectable ClpM protein was observed. Multiple analyses ruled out apicoplast-dependent instability, ER-associated degradation, misfolding or membrane sequestration. Attempts to express clpM or other plastid-derived genes using endogenous sequences were found to be toxic, suggesting nucleotide-sequence incompatibility. In contrast, a transgene carrying a second copy of the nuclear ClpC ortholog was readily expressed. Comparative analysis of ClpM and ClpC domain architecture showed that their ATPase domains form distinct evolutionary clusters, suggesting conserved but functionally divergent roles. Subsequently, domain-swap experiments between ClpC and ClpM rescued partial expression and identified specific domains as contributors to the nuclear-expression barrier. Together, these findings demonstrate that clpM retention in the apicoplast genome is enforced by multilayered constraints involving evolutionary conservation, nucleotide-sequence incompatibility, transcriptional block and protein-intrinsic translational barriers. This work provides experimental evidence for mechanisms that restrict organelle-to-nucleus gene transfer and contribute to organelle genome retention.

Pseudomonas aeruginosa is a globally recognized pathogen causing pulmonary, skin, and severe corneal infections (keratitis), with the potential to induce irreversible blindness if untreated. Spatial transcriptomic analysis of P. aeruginosa infected corneas identified elevated expression of the outer membrane proteins OprF and OprL and PA1414, which encodes the small RNA SicX in the corneal stroma compared with corneal epithelium. Comparative spatial transcriptomics analysis of corneas infected with an oprF transposon (TN) mutant showed reduced expression of the type III effector protein ExoT, which was absent in an oprF deficient mutant ({Delta}oprF) and in contrast to PA14, did not inhibit reactive oxygen species (ROS) production by neutrophils. Corneal infection with the {Delta}oprF mutant resulted in reduced corneal virulence and lower CFU compared to the parental PA14 strain. Collectively, our findings demonstrate a coordinated virulence program connecting OprF functionality with the release of ExoT and its ability to block ROS production and survive in infected corneas.