PLOS Pathogens

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease in which effective immunity requires sufficient inflammation to control parasites while limiting immune-mediated tissue damage. Transforming growth factor-beta (TGF{beta}) is an important regulator of immune homeostasis and has been implicated in VL, but how it directly controls parasite-specific CD4 T cell responses remains poorly understood. We used complementary transgenic mouse models with either enhanced or ablated TGF{beta} signalling in T cells during Leishmania donovani infection, combined with adoptive co-transfer of parasite-specific CD4 T cells to distinguish cell-intrinsic effects. Enhanced TGF{beta} signalling impaired hepatic parasite control and suppressed CD4 T cell immunity, reducing T helper 1 (Th1) cell differentiation, proliferation, accumulation of antigen-experienced cells, and expression of cytolytic molecules. Conversely, ablation of TGF{beta} signalling improved parasite control and promoted CD4 T cell expansion and Th1 cell differentiation, while increasing expression of cytolytic molecules and reducing interleukin-10-producing type 1 regulatory T (Tr1) cells. Adoptive co-transfer experiments confirmed that TGF{beta} directly restrained the expansion and Th1 cell differentiation of parasite-specific CD4 T cells and their acquisition of cytolytic features. Loss of signalling also impaired development of Tr1 cells and reduced expression of several chemokine receptors and co-inhibitory molecules associated with their regulatory function. However, enhanced signalling did not increase Tr1 cell development, indicating that the relationship between TGF{beta} signalling and immune regulation is not linear. TGF{beta} is a key cell-intrinsic regulator of CD4 T cell fate during experimental VL. Rather than acting solely as a general suppressor of inflammation, it calibrates the balance between protective and regulatory immunity by controlling CD4 T cell expansion, differentiation and effector function. Author summaryVisceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania parasites. The immune system must generate a strong enough response to control these parasites while preventing excessive inflammation that can damage tissues. We investigated how transforming growth factor-beta (TGF{beta}), an important regulator of immune responses, helps maintain this balance. Using mice in which signalling by TGF{beta} was either increased or removed specifically in T cells, we found that this pathway strongly influenced the development and function of CD4 T cells during infection. Increasing signalling suppressed the expansion of these cells and their development into inflammatory cells associated with parasite control. In contrast, removing signalling enhanced these responses and improved early parasite control, but also reduced the development of regulatory T cells that can limit inflammation. By studying parasite-specific T cells directly, we showed that many of these effects resulted from TGF{beta} acting within the T cells themselves. Our findings show that TGF{beta} does more than simply suppress immunity during VL. It helps determine the balance between CD4 T cell responses that control parasites and those that regulate inflammation, providing new insight into how immunity is shaped during chronic infection.

Protistan parasites of the genus Leishmania, infamous human and animal pathogens, can themselves be infected by endosymbiotic viruses, exemplified by Leishmania RNA viruses (LRVs). These viruses affect immune responses in vertebrate hosts and have been associated with adverse treatment outcomes. How parasites control replication of these viruses is not known. Intriguingly, functional RNA interference (RNAi) pathways that have been associated with antiviral responses across eukaryotes, are retained only in some Leishmania spp., including those of the subgenus Viannia. Here, we investigated effectors in the canonical RNAi response and the Piwi protein of the human pathogen L. (Viannia) guyanensis by gene ablation and identified Dicer-like 1 and Argonaute 1 proteins of the canonical RNAi pathway as critical for controlling viral RNA levels. Notably, we characterized virus-derived small interfering RNA (vsiRNA) levels and their unique properties including terminal modifications as well as, unusual for canonical Dicer cleavage, predominant perfectly matching sequence overlaps in blunt ended vsiRNA duplexes. Taken together, the data suggests that control of viral replication is directly mediated by the canonical RNAi response. This study opens the door to further investigations of antiviral RNAi in other protistan parasites and suggests that, where present, canonical RNAi is critical for such activities. Author summaryLeishmania parasites of humans and animals harbor endosymbiotic viruses, which, in some cases, have been shown to affect vertebrate immune responses and impact treatment. Thus, understanding how viral levels are controlled is critical to identify antiviral effectors, which, in turn, will allow studies on how viral levels impact parasite biology. Here, we investigated RNA interference pathways against its virus of the family Pseudototiviridae in a New World human pathogen L. guyanensis. To do that, we have produced and analyzed genetic knockouts of Dicer-like and Argonaute proteins involved in antiviral small RNA response. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/743808v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@cffe40org.highwire.dtl.DTLVardef@13d4012org.highwire.dtl.DTLVardef@395e60org.highwire.dtl.DTLVardef@631fed_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cathelicidins are a class of antimicrobial peptides (AMPs) that are part of the first line of defense of the innate immune system. While cathelicidin-derived peptides such as LL-37 can be directly bactericidal, Streptococcus pyogenes (Spy; Group A Streptococcus) is highly resistant to killing. Furthermore, Spy detects LL-37 through the CovRS two-component system to regulate its virulence factors. One effect of this signaling is the repression of expression of the bacterial protease SpeB. Prior work has also shown that SpeB, along with other bacterial proteases can cleave LL-37. However, it is unclear if SpeB cleavage of LL-37 impacts antimicrobial function and CovRS signaling activity. Using a genetic approach, we show that the presence SpeB did not significantly impact the killing of Spy by LL-37 relative to other known resistance factors. Furthermore, while SpeB cleaves LL-37, CovRS maintains sensitivity to LL-37 fragments. These results indicate that SpeB cleavage of LL-37 does not negatively impact virulence factor regulation in Spy.

Mareks disease virus (MDV) is an oncogenic herpesvirus that establishes latency in CD4 T cells, from which it reactivates to initiate productive replication and dissemination. To define host mechanisms governing the transition from latency to early and late lytic replication, we developed recombinant MDV expressing early RLORF4mRFP and late UL47eGFP, which was used to isolate latent (Lo; mRFP low), early lytic (Hi; mRFP high), and late lytic (DP; mRFPeGFP) populations from MDV-induced lymphoblastoid cell lines (LCLs). Two independent LCLs (Lines 62 and 82) were subjected to Illumina RNA sequencing after sorting-purified populations. Viral transcription increased progressively from Lo to Hi to DP populations; however, initiation of viral gene expression differed markedly between cell lines. During the early transition (Lo to Hi), Line 82 exhibited robust induction of 17 viral genes, whereas Line 62 showed only two differentially expressed viral genes. However, both lines converged on a highly conserved transcriptional program during the transition from latency (Lo) to late-lytic replication (DP), sharing 118 viral transcripts, most of which were structural and assembly-associated genes. Among the host transcriptional responses, Line 82 exhibited activation of TP53-associated stress signaling, chromatin remodeling factors, and RNA biogenesis pathways, consistent with a permissive cellular state. In contrast, Line 62 displayed enhanced inflammatory, metabolic, and proteostasis-associated signatures, suggesting a restrictive environment that limits early viral induction. Collectively, these findings demonstrate that MDV reactivation is a host-gated process in which the cellular state governs the initiation of the lytic switch, whereas downstream replication proceeds through a conserved viral program. IMPORTANCEThis study provides critical insights into the host-virus dynamics governing herpesvirus reactivation from latency. By developing a dual-reporter system using recombinant Mareks disease virus (MDV) in MDV-transformed lymphoblastoid cell lines (LCLs), an oncogenic herpesvirus model, reactivating cells were sorted for latent, early-lytic, and late-lytic populations from two independent LCLs. The initial transition from latency to early-lytic replication was highly variable and strongly influenced by cellular state: one line showed rapid viral gene induction linked to stress, chromatin remodeling, and permissive conditions, while the other exhibited a more restrictive, inflammatory/metabolic response. However, progression to late-lytic replication converged on a highly conserved viral transcriptional program dominated by structural genes. These findings establish that host cellular context gates the lytic switch in herpesvirus reactivation, offering a powerful framework for understanding latency control across oncogenic herpesviruses and potential therapeutic targets to prevent reactivation and tumor formation.

Streptococcus pneumoniae (pneumococcus) are asymptomatic colonizers of the nasopharynx but can progress to pulmonary and systemic pathogens upon influenza A virus (IAV) infection. Polymorphonuclear cells (PMNs) are required to control bacterial numbers, but we previously found that IAV infection impairs their ability to kill S. pneumoniae. Here, using a model that allows transition of pneumococci from colonizers to disease-causing pathogens upon IAV co-infection, we examined the signaling pathways impairing PMN responses. When we investigated the effect of type I interferons (IFN) produced upon IAV infection on PMN antibacterial activity, we found that PMNs treated with IFN were unable to kill S. pneumoniae ex vivo, and that in vivo blocking of IFN receptor 1 (IFNAR1) in IAV infected mice rescued PMN antibacterial function. In exploring what controls PMN responsiveness to IFN, we examined CD73, an ectonucleotidase that is known to regulate PMN function in primary pneumococcal pneumonia. To test if there is an intersection between CD73 and IFN signaling, we examined receptor levels and IFN production in wildtype versus CD73KO mice and found no difference in IFNAR expression on PMNs or IFN[a] and IFN levels in the lungs and circulation. However, CD73KO PMNs expressed significantly lower levels of the interferon stimulated protein IFIT1. When we looked at ex vivo PMN responsiveness to IFNs, CD73KO PMNs were less responsive to IFN-mediated inhibition of antimicrobial activity. In exploring mechanisms, we found that CD73 expressing PMNs had elevated production of reactive oxygen species in response to IAV challenge, that paradoxically impaired their ability to kill S. pneumoniae. Importantly, despite similar pathogen loads in the respiratory tract, co-infected CD73KO mice cleared bacteremia and survived significantly better than wildtype controls. These findings suggest that CD73 impairs host defense against IAV/S. pneumoniae co-infection in part by sensitizing PMNs to type I IFN-mediated inhibition of antibacterial function. Author SummaryDespite available therapeutics and vaccines, secondary bacterial pneumonia following influenza A virus (IAV) infection remains a major cause of disease. A common cause of secondary bacterial pneumonia are Streptococcus pneumoniae (pneumococcus), bacteria that resides asymptomatically in the nasopharynx, but upon viral infection can transition to cause severe disease in susceptible hosts. In this study we examined how host responses change during single versus polymicrobial infections. We focused on neutrophils, which are innate immune cells that are required for effective clearance of S. pneumoniae, and proper control of IAV. We found that the immune response to IAV, mediated by type I interferons (IFN), impair the ability of neutrophils to kill bacteria. We identified an enzyme called CD73 to be required for the ability of neutrophils to respond to IFN. In exploring mechanisms, we found that IFN and CD73 result in dysregulated reactive oxygen species production by neutrophils. Importantly, this impairs the ability of the host to clear bacteria that spread from the lungs to the blood upon viral co-infection and results in overall worse host outcome. This study describes a novel interaction between CD73 and type I interferons and provides a new therapeutic target to treat secondary pneumococcal pneumonia.

Kaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that causes Kaposis sarcoma and several lymphoproliferative disorders. The KSHV open reading frame (ORF) 16 encodes viral Bcl-2 (vBcl-2), which inhibits apoptosis and autophagy. vBcl-2 is required for efficient lytic replication and production of infectious progeny; however, this essential role is independent of its established functions regulating cell death. To identify host factors that may contribute to vBcl-2 function, we employed proteomic analysis of HA-vBcl-2 immunoprecipitates from lytically reactivated KSHV-infected cells. This analysis identified the BTB-Kelch protein KLHL9, a substrate-specific adaptor for Cullin 3-RING ligase (CRL3) complex, as a candidate vBcl-2-associated factor. We validated the vBcl-2-KLHL9 association by reciprocal co-immunoprecipitation from infected cells, and by ectopic expression in uninfected cells. During lytic reactivation, KLHL9 redistributed from a predominantly cytoplasmic/perinuclear pattern to a mitochondrial pattern that overlapped with HA-vBcl-2. Alanine-scanning mutagenesis mapped a KLHL9-interaction determinant to the N-terminal region of vBcl-2, and vBcl-2 mutants defective in binding failed to colocalize with KLHL9. Conversely, KLHL9 deletion analysis implicated the C-terminal Kelch-repeat region in efficient association with vBcl-2. AlphaFold modeling supported the interface regions. However, vBcl-2 did not show detectable KLHL9-dependent ubiquitination, suggesting that it is unlikely to be a substrate of a KLHL9-containing complex. Functionally, CRISPR/Cas9-mediated KLHL9 disruption reduced lytic viral protein accumulation and infectious progeny production, while re-expression of sgRNA-resistant KLHL9 partially restored these phenotypes. Together, these findings suggest that vBcl-2 may engage host CRL3 complexes during productive infection, and identify KLHL9 as a vBcl-2-associated host factor that supports efficient KSHV lytic replication. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is a cancer-associated virus that alternates between a latent cycle and a productive lytic cycle, during which new infectious virus particles are made. The viral protein vBcl-2 is best known for regulating cell-death and autophagy pathways, but earlier studies showed that it is required for efficient virus production through additional, non-canonical functions. In this study, we identify the cellular protein KLHL9 as a host factor that associates with KSHV vBcl-2. KLHL9 normally functions as a specific adaptor in protein ubiquitination pathways that regulate the turnover, localization, or activity of target proteins through ubiquitination. We show that vBcl-2 and KLHL9 associate with one another, and that KLHL9 redistributes toward the mitochondria in cells expressing vBcl-2. Importantly, loss of KLHL9 impairs the expression of viral lytic proteins and reduces the production of new infectious viruses. This defect resembles the phenotype caused by loss of vBcl-2, suggesting that KSHV employs vBcl-2 to engage KLHL9-dependent host machinery during productive infection. Understanding this interaction may help reveal how herpesviruses reprogram infected cells to create an environment that supports virus production.

Legionella pneumophila (L. pneumophila) is the causative agent of Legionnaires' disease, a severe bacterial pneumonia. Difficulty in diagnosing Legionnaires' disease leads to an underreporting of cases and delayed treatment. Rapid-acting, broad-spectrum therapies are needed to treat pathology while avoiding antibiotic resistance. We showed that gelsolin knockout (gsn-/-) mice succumb more quickly to severe L. pneumophila infection despite no difference in bacterial loads in the lung compared to wild type mice. There is an increase in CXCL1/KC production from macrophages from gsn-/- mice, which is accompanied by increased neutrophils and apoptosis in their lungs. Neutrophils lacking gelsolin produce fewer neutrophil extracellular traps, and their mitochondrial capacity is diminished in response to L. pneumophila. Gelsolin is required for maintaining mitochondrial network morphology and respiration in L. pneumophila infected macrophages. When given recombinant gelsolin protein, gsn-/- mice survive significantly longer during severe L. pneumophila infection, with reduced lung pathology, and the inflammatory signature of their macrophages was reduced in vitro. Together, gelsolin protects mice during severe L. pneumophila infection, dampens inflammation, promotes mitochondrial health, and maintains neutrophil function.

RNA interference (RNAi) is a central antiviral defense mechanism in fungi, yet relatively few mycoviral suppressors of RNA silencing (VSRs) have been functionally characterized, particularly in phytopathogenic hosts. Botrytis virus X (BVX), a positive-sense RNA virus in the family Alphaflexiviridae, infects Botrytis cinerea and encodes five predicted open reading frames (ORFs), most of which have unknown functions. Here, we screened BVX ORFs 2-5 for RNA silencing suppressor activity using complementary GFP-based assays in Nicotiana benthamiana and examined the leading candidate in the fungal host B. cinerea. BVX ORF2 (X2) enhanced GFP transcript and protein accumulation in assays where silencing is triggered by sense RNA but failed to suppress silencing triggered by hairpin-derived siRNAs or miRNA-guided targeting, indicating a trigger-restricted suppressor phenotype. In B. cinerea, transgenic expression of X2 was associated with reduced induction of the RNAi associated genes BcDCL1 and BcDCL2 compared to empty vector controls, with the strongest effect observed on BcDCL1. In a virus-infected fungal background, X2 expression was also associated with increased viral RNA accumulation. Together, these results identify BVX X2 as a BVX-encoded, trigger-restricted suppressor of RNA silencing and link its expression to altered RNAi-related gene induction and increased viral RNA accumulation in B. cinerea.

Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) nonstructural protein 1 (Nsp1) dampens the host immune response by shutting off host gene expression, a strategy that has remained evolutionary conserved among a diverse range of coronaviruses (CoVs). Residues important for SARS-CoV-2 Nsp1 mediated host shutoff have been incompletely defined. We have generated and characterized the ability of four Nsp1 mutants to inhibit host gene expression in both plasmid-based overexpression assays and utilized reverse genetic approaches to generate recombinant (r)SARS-CoV-2 expressing each Nsp1 mutant to investigate their impact on viral infection. Infection of K18-hACE2 transgenic mice with the rSARS-CoV-2 Nsp1 mutants resulted in reduced pathogenicity as determined by body weight maintenance and survival, attenuated viral replication in lung and nasal turbinate, distinct immune signatures in lung, and less severe lung pathology in comparison to wild-type (WT) virus-infected mice. Our data suggests amino acid residues in the C-terminal domain, in addition to the linker domain, of SARS-CoV-2 Nsp1 are critical determinants of viral pathogenicity as a result of their role in disrupting host gene expression. The reduced pathogenicity of rSARS-CoV-2 Nsp1 mutants highlights the potential for targeting Nsp1 for rational design of viral inhibitors and development of live-attenuated vaccine strategies as effective prophylactic and therapeutic treatments, respectively, to combat SARS-CoV-2 and possibly other CoV infections. IMPORTANCETo mitigate the ongoing public health threat posed by Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) and prepare for future coronavirus (CoV) outbreaks, there is an urgent need for effective prophylactic and therapeutic strategies, including vaccines and antivirals. The nonstructural protein 1 (Nsp1) is a conserved CoV virulence factor that suppresses host gene expression and disrupts immune responses. However, the contribution of specific Nsp1 residues to CoV pathogenesis remains unclear. Here, we identify residues within the C-terminal and linker regions of Nsp1 as critical determinants of SARS-CoV-2 pathogenicity in vivo. These findings advance our understanding of CoV host shutoff mechanisms and support Nsp1 as a promising target for the development of live-attenuated vaccines and antiviral therapeutics.

Severe dengue can damage the liver through unestablished mechanisms. We investigated the role of myeloperoxidase (MPO), a neutrophil enzyme, in dengue through patients, fatal liver samples, and mouse infection models. Observations from two independent clinical cohorts revealed elevated plasma MPO levels in dengue and, in one cohort, MPO was further linked to liver injury markers during the critical phase of disease, whereas livers from dengue fatal cases revealed MPO build-up in the vicinity of CD177+ activated neutrophils. In mice, dengue led to MPO overexpression, oxidative damage, and broad activation of innate and systemic inflammatory pathways in livers. Blocking MPO activity alleviated these and improved survival in one model and delayed disease progression without preventing death in another. These findings establish MPO as a functional mediator of severe dengue-associated liver injury and inflammation, which warrants further preclinical investigation into its hepatic pathogenic mechanism and its validity as target for therapeutic intervention.

Certain strains of the facultative intracellular bacterial pathogen Listeria monocytogenes are thought to invade cranial nerves in the gut and disseminate directly to the brainstem to cause rhombencephalitis in both humans and ruminants. Bacteria with actin tails were previously observed within neurons of naturally infected sheep, but the mechanism for how these neurotropic strains access the nervous system has not been well characterized. Using a foodborne mouse model of listeriosis, we show here that bypassing the gut phase of infection prevents colonization of the brain, confirming that invasion of the nervous system happens in the intestinal tract. L. monocytogenes did not efficiently invade neuronal cell lines, although they could replicate exponentially in the cytosol and form actin tails. Instead, the neurotropic strains displayed a preferential ability to invade enteric glial cells, a specialized subset of glia that support neurons and are critical for intestinal homeostasis. Using an in vitro co-culture system, we demonstrated that neurotropic L. monocytogenes could readily invade enteric glial cells and use ActA-mediated actin-based motility to spread to adjacent neurons. These results suggest that invasion of enteric glial cells is a novel virulence strategy that can promote brainstem infection following foodborne transmission of L. monocytogenes. IMPORTANCEThis study provides further evidence for dissemination of neurotropic strains of L. monocytogenes from the gut directly to the brain via axonal migration using foodborne mouse model of listeriosis. It is the first report showing that enteric glial cells, a specialized subset of cells in the gut that support intestinal neurons, are susceptible to pathogenic bacterial infection.

Antigenic variation in Trypanosoma brucei relies on strict monoallelic expression of variant surface glycoprotein (VSG) genes from a single telomeric expression site (ES), a process sustained by the extranucleolar RNA polymerase I (Pol I) transcriptional body known as the expression site body (ESB). Although the ESB is essential for VSG expression, the mechanisms governing its assembly and maintenance remain poorly understood. Here, we identify SUMOylation as a central regulator of ESB organization and demonstrate that the balance between SUMO conjugation and deconjugation determines the transcriptional state of VSG expression sites. Ectopic expression of the SUMO protease TbSENP disrupted the highly SUMOylated nuclear focus associated with the active-ES, displaced Pol I from its extranucleolar compartment, and markedly increased VSG in situ switching frequency, indicating that continuous SUMOylation is required to preserve ESB integrity. Conversely, targeted recruitment of the SUMO-conjugating enzyme TbUBC9 to a silent ES locally restored SUMOylation, induced de novo formation of an extranucleolar Pol I compartment, activated transcription of the corresponding telomeric VSG gene, and generated stable antigenic switchers expressing the new surface coat. Local SUMOylation preceded Pol I redistribution, supporting a model in which SUMO-dependent interactions nucleate assembly of a transcriptionally competent ESB. Together, our findings identify SUMOylation as both a structural and regulatory determinant of nuclear organization in T. brucei and suggest that dynamic SUMO homeostasis governs the assembly, maintenance, and remodeling of this specialized transcriptional body. Significance StatementAntigenic variation in Trypanosoma brucei depends on the monoallelic expression of Variant Surface Glycoprotein (VSG) genes from a specialized RNA polymerase I transcriptional compartment known as the Expression Site Body (ESB). However, the molecular signals that govern transitions between active and silent expression sites have remained unknown. We show that SUMOylation acts as a reversible molecular switch: disruption of SUMO homeostasis dismantles ESB organization and promotes VSG switching, whereas localized SUMOylation is sufficient to nucleate a functional transcriptional compartment and activate a silent VSG expression site. Our findings establish SUMOylation as a central regulator of nuclear architecture and antigenic variation.

Intracellular pathogens localize to various niches in the host cells to avoid immune detection. However, very little is known about bacteria that enter the host nuclei. Here we report that Burkholderia thailandensis, a facultative intracellular pathogen, can enter eukaryotic nuclei and replicate. Nuclear invasion events were rare, occurring 1 in 500-1,000 infected cells, and inhibition of cell division further reduced the frequency of these events. Moreover, we show that nuclear entry requires actin tail motility, although it is independent of other virulence factors such as the Type III Secretion System, Type VI Secretion System-5, and flagella motility. Inactivation of actin tail motility by deleting bimA or inhibition of actin polymerisation by cytochalasin D abolished nuclear entry. Surprisingly, we observed that accumulation of B. thailandensis in the nucleus activated assembly of the Type VI Secretion Systems-5. We further show that Shigella flexneri also enters nucleus in an actin polymerization dependent mechanism. Together, we show that actin tail forming intracellular pathogens occasionally localize to the nucleus, and while this largely requires host cell division, it may provide pathogens with a protective niche in certain mitotically active cells, such as skin, gut or epithelial cells.

Human -defensins can paradoxically inhibit or enhance human adenovirus (HAdV) infection. Defensin-resistant HAdVs exploit -defensins as molecular bridges to bypass canonical receptors, but whether this mode of entry redirects intracellular trafficking or contributes to defensin resistance remains unknown. Here we systematically compared intracellular trafficking after canonical receptor-mediated versus defensin-mediated entry using a defensin-resistant chimeric virus based on HAdV-C5 together with the human -defensins, HD5 and HNP1. Pharmacological perturbations, capsid mutants, and imaging revealed that both entry routes rapidly converge on common checkpoints. Defensin-mediated infection showed the same sensitivity to endosomal acidification blockade and comparable co-localization with early endosomes as canonical entry. Notably, the membrane-lytic activity of HD5 did not substitute for viral protein VI, indicating that endosomal escape still requires intact protein VI function. Downstream transport also overlapped, as defensin-mediated infection, like canonical entry, depended on dynein-driven microtubule transport and passage through the microtubule-organizing center (MTOC) for nuclear entry. Co-infection experiments further showed that defensin-sensitive and defensin-resistant viruses retain their intrinsic phenotypes within the same cell, indicating that defensin effects are virion-autonomous and determined at or before cell entry rather than by downstream trafficking. These findings support a model in which resistance to -defensin neutralization is governed by capsid features that control defensin binding and its effects on uncoating, not by access to an alternative intracellular pathway. Author SummaryHuman adenoviruses can be neutralized by -defensins, antimicrobial peptides that bind the capsid and block uncoating. Paradoxically, some defensin-resistant adenoviruses infect cells more efficiently in the presence of defensins, with the peptides acting as molecular bridges that allow the virus to attach independently of its normal receptors. We asked whether this alternative attachment route changes how adenoviruses traffic through the cell, which could contribute to defensin resistance. Using labeled viruses, inhibitors, capsid mutants, and receptor knockout cells, we compared trafficking after canonical versus defensin-mediated entry. Both routes converged at the earliest steps of infection, with identical endosomal acidification requirements, similar early endosome colocalization, and the same dependence on microtubule transport to the nucleus. Co-infection experiments demonstrated that sensitive and resistant viruses retained distinct phenotypes in the same cell, indicating susceptibility is set at or before entry. These results demonstrate that defensin effects are decided at the plasma membrane, where capsid features, not alternative entry routes, drive resistance. Adenovirus entry is flexible at the cell surface, where the virus can attach through multiple mechanisms, but narrows to a single, constrained trafficking route inside the cell. Our findings inform how defensin-resistant vectors may behave in defensin-rich tissues during gene therapy or virotherapy.

Many pathogens actively suppress early host immune responses to enhance their fitness. Mitochondria function as key regulators of immune activation, yet whether pathogens suppress host immunity by manipulating mitochondrial metabolism in vivo remains largely unknown. During an innate immune response, the metabolite itaconate increases in abundance and acts as an immunomodulator, due to its inhibition of succinate dehydrogenase (SDH), a key mitochondrial regulator of cellular immunity. We hypothesized that Mycoplasma gallisepticum (MG), a recently emerged pathogen of wild songbirds, most notably house finches (Haemorhous mexicanus), suppresses early host immune responses by limiting SDH-dependent immune activation via itaconate. We tested these hypotheses using experimental 3-day infection of finches with heat-killed MG, live MG or pharmacological elevation of the SDH inhibitor itaconate. Following inoculation, we quantified intracellular itaconate and mitochondrial respiratory function in peripheral blood mononuclear cells (PBMCs) and pro-inflammatory cytokine gene expression in erythrocytes, in addition to infected tissues (trachea and conjunctiva). Heat-killed MG increased SDH-dependent mitochondrial respiration in PBMCs and cytokine gene expression in erythrocytes, but live MG did not show these increases, but revealed increased itaconate accumulation in PBMCs. Dimethyl itaconate administration reproduced the suppressed metabolic and immune phenotype in blood cells observed with live MG, suggesting an itaconate-associated mechanism. In contrast, live MG increased mitochondrial respiration and gene expression levels of cytokines in eyelid conjunctiva, whereas other treatments did not. These findings indicate that MG suppresses host metabolic and cytokine signaling in systemically circulating immune cells through a mechanism consistent with itaconate-mediated inhibition of SDH-dependent mitochondrial respiration, while still inducing an inflammatory response at the site of infection. Our data suggest that MG, like other pathogens, can commandeer host immunometabolic pathways during infection to their benefit and that mitochondria are a key site of competition between host and pathogen.

Host cell invasion by malaria parasites requires specific molecular interactions with host receptors. Plasmodium vivax merozoite invasion of reticulocytes is mediated by P. vivax Duffy binding protein (PvDBP) and its homolog, P. vivax erythrocyte binding protein (PvEBP). Here, we identify and characterize two novel P. vivax merozoite proteins, PvMP45 and PvMP36, which co- localize with PvDBP and PvEBP in the micronemes and bind reticulocyte receptors. PvMP45 and PvMP36 share high sequence identity with their P. knowlesi homologs, PkMP45 and PkMP36, which form a complex with other invasion related proteins. Field studies reveal that naturally acquired antibodies against PvMP36, PvEBP and PvDBP are associated with protection against clinical P. vivax malaria. We demonstrate that naturally acquired antibodies to PvEBP bind Fcy receptors and likely mediate protection by enabling opsonic phagocytosis. In addition, we show that combining antibodies against PvDBP and PvMP36 results in an additive invasion inhibitory effect against P. vivax blood stages. These results suggest that combining PvDBP, PvEBP and PvMP36 in a multivalent blood stage vaccine could elicit diverse immune mechanisms against P. vivax to achieve high efficacy. ImportanceAll the clinical symptoms of malaria are attributed to the blood stage of malaria parasites during which merozoites invade and multiply within red blood cells. A clear understanding of the host- parasite interactions that enable invasion can open paths for development of novel methods to block parasite growth and prevent malaria. Here, we identify and characterize two novel invasion related proteins from P. vivax merozoites that form an invasion complex and bind host RBC receptors. We demonstrate that antibodies targeting these proteins can block RBC invasion by P. vivax and naturally acquired antibodies that develop following P. vivax infection against one of these proteins are associated with protection against P. vivax malaria. These studies not only expand our understanding of the molecular mechanisms that enable host cell invasion by P. vivax but open new avenues for development of vaccines to protect against P. vivax malaria.

With the rise of antimicrobial resistance, anti-virulence therapeutics are a viable alternative to circumvent resistance pressures. Hypothetical genes and proteins are an under-studied source of potential virulence factor targets. We performed bioinformatic analyses to identify conserved hypothetical genes enriched in pathogenic Pseudomonas aeruginosa but not in non-pathogenic strains. This analysis identified an atypical BlaIR system, which we named pvmSR, that regulated P. aeruginosa virulence in a Caenorhabditis elegans infection model. This is in contrast with the typical BlaIR system from Staphylococcus aureus, which regulates resistance to {beta}-lac-tam antibiotics. The{Delta} pvmSR mutant showed reduced virulence in a C. elegans slow-killing assay. To understand how PvmSR regulated virulence in vivo, we performed dual RNA-seq to analyze transcriptomic changes in both C. elegans and P. aeruginosa. We found that C. elegans responded to P. aeruginosa {Delta}pvmSR infection by decreasing expression of lysosome and phagocytosis pathways. In P. aeruginosa {Delta}pvmSR, we observed decreased gene expression of several known virulence factors including the hydrogen cyanide synthase, hcnC, and heparinase, hepP. Additionally, we observed dysregulation in genes important for quorum sensing and biofilm formation. Collectively, our findings indicated that PvmSR contributed to virulence regulation and may serve as a potential anti-virulence target.

Western flower thrips (WFTs) are critical vectors of tomato spotted wilt virus (TSWV), transmitting it via a circulative-propagative cycle. The insect-virus relationship is unusual in that only larvae can acquire the virus for transmission to plants to occur. During the larval stage, the virus circulates and replicates within many organs, reaching the salivary glands before the insect pupates, and remaining in infected organs when the insect becomes an adult. The virus continues to replicate in the salivary glands of adult insects, after which it is inoculated into plants via saliva during feeding. Understanding the interactions between TSWV and the WFT salivary glands is critical to furthering investigations of TSWV inoculation and efforts to block the spread of this devastating plant virus. Here, we document transcriptomic changes associated with TSWV infection of the salivary glands of adults (males and females) and second instar larvae. Gene sets enriched in adult male, female, and larval genes revealed a core set of genes associated with WFT salivary glands, as well as genes that differed between sexes and between adults and larvae. The transcriptome response to TSWV infection was higher in larvae (second instar in this study) than in adults, with nearly a 10x increase in differentially expressed genes. We hypothesize this occurred because larvae efficiently acquire the virus and the virus first enters the SGs at the L2 stage, whereas adult SGs are infected only if acquisition occurred in the larval stage. Thus, assessment of larvae detects responses to the early stages of infection, while assessment of adults detects responses to the later stages of infection. Similarly, functional changes in larval salivary glands were more diverse, with significant transcriptome differences associated with growth and development in this tissue during infection. Lastly, a comparative analysis of changes in a published SG proteome revealed a correlation between transcript and protein levels during infection, but little overlap between significant TSWV-responsive transcripts and proteins. These studies provide critical insight into the molecular changes associated with the first breach of the SGs in larvae by TSWV, revealing a markedly different transcriptomic response compared to that in adults.

Helicobacter pylori strains encoding the cag-pathogenicity island (cag-PAI) and the effector toxin cagA are associated with worse disease outcomes. The cag-PAI encodes the Cag type IV secretion system (Cag-T4SS) which injects CagA and other bacterial products into gastric epithelial cells. Prior work revealed that host adaptive immunity promotes recombination in the cag-PAI gene cagY to attenuate Cag-T4SS activity during chronic infection, suggesting a fitness cost to assembling an active Cag-T4SS. To explore potential selective benefits and costs for the Cag-T4SS and CagA, we employed single strain and competitive infections at both acute and chronic timepoints in wildtype mice and transgenic mice that either attenuate innate immune responses or promote gastric pathology independent of H. pylori infection to examine the relative fitness of mutant H. pylori strains. Our results suggest that an active Cag-T4SS and CagA confer a fitness benefit during initial colonization through Cag-T4SS activity-dependent epithelial cell interactions that activate cancer-related signaling pathways. However, increasing gastric inflammation confers a fitness cost to CagA translocation, promoting Cag-T4SS shutoff. Targeted and whole genome sequencing revealed multiple mechanisms of Cag-T4SS attenuation, with recombination-mediated changes in cagY prevalent at early timepoints and mutations in a variety of Cag-T4SS structural genes accumulating as disease progresses. The need for Cag-T4SS activity and CagA translocation during initial gland colonization likely underlies the mutational pattern observed. Collectively this work reveals new insights into selective constraints on the H. pylori Cag-T4SS as well as resultant genetic adaptation processes that lead to retention of the cag-PAI and virulence.

Mycoviruses modulate fungal fitness and pathogenicity, yet their biological roles in obligate biotrophic phytopathogens remain poorly understood. Here, we report the first functional characterization of totiviruses in rust fungi, identifying five novel totiviruses, designated Puccinia triticina totivirus 1 to 5 (PtTV1?PtTV5), from the wheat leaf rust fungus Puccinia triticina (Pt). All five PtTVs possess the canonical genomic architecture of Totiviridae, including two overlapping ORFs and a conserved ?1 ribosomal frameshifting motif. Transcriptional profiling revealed that PtTVs are highly expressed during early Pt infection. PtTV-encoded proteins suppressed BAX-triggered programmed cell death in Nicotiana benthamiana, indicating immune-suppressive activity. Using BSMV-mediated host-induced gene silencing (HIGS), we showed that knockdown of PtTV transcripts significantly impaired fungal hyphal expansion and uredinial formation, concomitant with enhanced host H?O? accumulation. A survey of 90 Pt field isolates from four major wheat-growing regions of China revealed that PtTVs are ubiquitously distributed in natural rust populations. Collectively, these findings demonstrate that totiviruses function as virulence-promoting symbionts in Pt, establishing for the first time a functional link between totiviral infection and enhanced pathogenicity in cereal rust fungi and identifying candidate targets for RNAi-based disease control.