Virulence

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CMank13) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22-up and tsa56-down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl2, the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CMank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen. IMPORTANCEOrientia tsutsugamushi causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease, Orientia is the only one for which genetic tools have not been developed. This has limited understanding of O. tsutsugamushi-host dynamics that drive the bacteriums pathobiology and hindered development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in O. tsutsugamushi. Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for O. tsutsugamushi.

The pathogenic fungus Trichosporon asahii causes severe mycoses in immunocompromised hosts, such as neutropenic patients. In Cryptococcus neoformans, the unfolded protein response (UPR) sensor Ire1 induces hxl1 mRNA splicing and contributes to stress responses and virulence. The function of Ire1-triggered hxl1 mRNA splicing in stress tolerance and virulence of T. asahii, however, remains unclear. Here, we demonstrated that ire1- and hxl1 gene-deficient T. asahii mutants are sensitive to dithiothreitol (DTT), an inducer of endoplasmic reticulum stress, and exhibit reduced virulence in a silkworm infection model. DTT treatment induced hxl1 mRNA splicing in the wild-type strain, whereas ire1 gene-deficient mutants did not undergo hxl1 mRNA splicing. The ire1 gene-deficient mutants were more sensitive than the parent strain to DTT, H2O2, Congo red, and SDS, and showed reduced virulence in silkworms. Similarly, hxl1 gene-deficient mutants exhibited increased sensitivity to these stressors and reduced virulence. Both the ire1 gene-deficient and hxl1 gene-deficient mutants showed decreased expression of reactive oxygen species-detoxifying related genes CAT2, SOD1, and SOD2, compared with the parent strain. Together, these findings suggest that Ire1-triggered hxl1 mRNA splicing contributes to stress resistance and virulence in T. asahii.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.

Lyme arthritis, a manifestation of Lyme disease, is triggered by the spirochetal bacterium Borrelia burgdorferi (Bb), which is transmitted through the bite of the Ixodes tick. Although multiple studies have been conducted on the complex host immune response in Lyme arthritis, the spatial gene expression environment in the joint tissue remains unexplored. Here, we applied spatial transcriptomics to ankle joints of C3H mice infected with Bb, profiling tissues at peak inflammation (two weeks post infection) and after antibiotics (four weeks post-infection) during inflammation resolution. Analysis revealed spatially restricted signatures: pro-inflammatory responses dominated synovial and fibroblast populations two weeks post-infection, with elevated levels of Vimentin and I-Ek gene - and Vimentin protein - expression localized to these regions. By four weeks post-infection during the inflammation resolution phase, levels of Vimentin and I-Ek related gene and protein expression were reduced. Further, we noted an increase in the CD54+ and CD106+ double-positive population in infected mice joints compared to the vehicle treated controls. Notably, fibroblasts and synoviocytes in the medial joint regions adopted immune-like phenotypes during peak inflammation, while the same cell types in the exterior humeroradial joint displayed a more infection-resilient phenotype. These spatially resolved maps demonstrate that joint microenvironments play a crucial role in pathogenesis, offering unique insights into Lyme arthritis pathology.

A low abundance or absence of protective lactobacilli during acute trichomoniasis is a well-known phenomenon that was reported in multiple studies and is the hallmark of a T. vaginalis (TV) infection. However, a crucial question that remains unanswered is whether alterations in the lactobacilli population precede TV infection or whether the parasite plays an active role in lactobacilli disappearance. Our findings showed that TV efficiently phagocytosed the dominant Lactobacillus species L. jensenii (LJ). Phagocytosis proceeds via a pseudopodia-independent mechanism reminiscent of sinking with a preference for viable cells. The presence of viable LJ leads to an increase in secretion of 27 TV proteins, including TvGH25 lysozyme. This enzyme cleaves peptidoglycan, a major component of the bacterial cell wall. TV overexpressing TvGH25 effectively lowers the bacterial cell count, evidencing the enzyme's antimicrobial potential. These data support the notion that TV cells can suppress the Lactobacillus population through a combination of targeted secretory response and phagocytic activity, revealing novel potential targets for developing alternative therapeutic strategies against trichomoniasis.

Gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, poses a growing global public health threat due to the rapid emergence of multidrug-resistant strains and the limited availability of effective treatments. Since there are no known lytic gonophages, we explored prophages present in the genome of N. gonorrhoeae FA1090, with a particular focus on prophage-encoded endolysins. In this study, we evaluate antigonococcal properties of prophage-encoded endopeptidases with the NlpC/P60 enzymatic domain. Recombinant endolysin Phi1gp518 exhibits intrinsic bactericidal activity against non-permeabilized N. gonorrhoeae FA1090 cells. Furthermore, it shows an expanded host range against clinical gonococcal isolates. The gonolysin remains stable across all human body temperatures, a pH range of 5-10, and shows no cytotoxic effects toward human cervical epithelial cells, supporting its potential safety for therapeutic applications. Additionally, Phi1gp518 impairs the formation of gonococcal microcolonies and prevents proper biofilm establishment. The antigonococcal properties of Phi1gp518 endopeptidase make it a good candidate for further protein engineering and development as an alternative treatment strategy for drug-resistant N. gonorrhoeae infections.

The recent shift in Bangladesh from tradition backyard rearing system to modern commercial layer farming has made the birds immune to infectious diseases. Pediculosis, however, continues to pose a challenge in modern production system due to its invasive nature, often going unnoticed and neglected as it is typically non-lethal, yet capable of causing significant production losses. Lice infestation is a persistent threat in poultry production; however, its implications in battery-caged commercial layer hens in Bangladesh remain insufficiently characterized. The study aimed to identify the causative louse species and evaluate its associations with clinical pathology, hematological alteration and productive performance in 30 white-feathered (15 infested + 15 non-infested) and 30 brown-feathered (15 infested + 15 non-infested) laying birds from two commercial farms in Tangail. Morphological characterization confirmed the parasite as Menacanthus stamineus, distinguished by a dorsoventrally flattened body, parabolicallly rounded head wider than long, concealed club-shaped antennae, an oblong-oval abdomen with fine setae and three pairs of short legs each bearing paired claws. Infested birds exhibited consistent clinical pathology, including pale combs, petechial hemorrhages around the vent, severe feather damage with alopecic and exudative areas and incidence of irregular and broken-shelled eggs. Production performance analysis revealed significant reduction in hen-day egg production, egg weight, and feed intake, accompanied by significantly increased feed conversion ratios. Hematological evaluation demonstrated significantly reduced hemoglobin concentration, hematocrit and erythrocyte counts in infested hens, indicating mild anemia and compromised oxygen-carrying capacity. Collectively, pediculosis was strongly associated with lice-induced self-inflicted injury and cannibalism, systemic physiological stress, impaired erythropoiesis, reduced production efficiency and compromised welfare in caged laying hens. To best of our knowledge, it was the first integrative reports from Bangladesh documenting M. stramineus infestation in battery-caged commercial layer system with concurrent evidence of hematological disruption and measurable productivity losses, underscoring its epidemiological and economic significance and urgent need for targeted, evidence-based ectoparasite control strategies.

Cryptococcus neoformans is the predominant causative agent of cryptococcal meningitis in immunocompromised individuals. Conversely in immunocompetent individuals, C. neoformans establishes a latent pulmonary infection characterized by a paucity of clinical symptoms. Using a mouse inhalation model of latent C. neoformans infection, we previously showed that CD4 T-cells are necessary for preventing fungal proliferation in the lungs. In the current study, we performed single cell RNA sequencing (scRNAseq) and found that the CD4 T-cell response was both highly heterogenous and dichotomous during pulmonary C. neoformans infection, with concomitant expression of genes related to Th1 polarization (Tbx21, Ifng) and immune regulation (Ctla4). First, we demonstrated that cells with Th1-like phenotypes are necessary and sufficient to control latent infection via adoptive transfer of T-bet positive cells into infection-matched CD4-depleted recipient mice. Second, scRNAseq analysis revealed the subpopulation of effector CD4 T-cells that co-expressed Ctla4 and Gata3 was significantly higher than a subpopulation that co-expressed Ctla4 and Tbx21. Furthermore, our data suggested that CTLA-4 upregulation is beneficial against C. neoformans infection, as CTLA-4 blockade promoted fungal proliferation. Thus, we propose a model wherein Th1 control of latent C. neoformans infection is supported by CTLA-4 suppression of detrimental Th2 activation.

Malaria, of which the most virulent form is caused by Plasmodium falciparum parasites, remains a major global health burden. The appearance of resistance to first line treatments artemisinin-based therapies, emphasizes the need to identify new parasite vulnerabilities to develop new therapeutics. Phosphoinositides are central regulators of membrane identity, vesicular trafficking, and signaling, and their synthesis depends on tightly controlled phosphatidylinositol transfer by Sec14-like phosphatidylinositol transfer proteins in many eukaryotes, yet their roles in P. falciparum remain poorly defined. Here, we analyzed six P. falciparum Sec14 domain-containing proteins: PfSec14-1 (PF3D7_0626400), PfSec14-2 (PF3D7_0629900), PfSec14-3 (PF3D7_0717100), PfSec14-4 (PF3D7_0920700), PfSec14-5 (PF3D7_1007200), and PfSec14-6 (PF3D7_1127600). Domain organization segregates these proteins into a BNIP-2 and Cdc42GAP homology (BCH) subfamily (PfSec14-3, PfSec14-5) and a canonical Sec14 subfamily (PfSec14-1, PfSec14-2, PfSec14-4, PfSec14-6). Yeast complementation assays showed that PfSec14-1, PfSec14-4, and PfSec14-6 partially rescue growth of a temperature-sensitive sec14 mutant, suggesting phosphatidylinositol and phosphatidylcholine transfer activity. Gene disruption revealed that PfSec14-1 is important for asexual blood-stage proliferation, whereas PfSec14-2 is dispensable under standard culture conditions. In contrast, mislocalization of PfSec14-1 and PfSec14-4 using a knock-sideways approach did not impair asexual growth. Subcellular localization indicates distinct distributions for PfSec14-1, PfSec14-2, and PfSec14-4. Together, these findings reveal functional and spatial diversification of Sec14-like phosphatidylinositol transfer proteins in P. falciparum.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm), remains a major burden to cattle health and the agricultural industry. Mmm is an atypical bacterial pathogen that appears to lack classical virulence factors that cause direct tissue injury (i.e. toxins), and little is known about the mechanisms driving its pathogenicity. The host immune response is believed to be implicated in CBPP pathology, though the molecular mechanisms underlying lesion initiation, progression and chronicity are poorly defined. Classical pathology describes a continuum of lung lesions starting from early inflammation to more mature necrotic lesions and formation of fibrotic sequestra. However, the host transcriptional response driving this potentially immunopathological progression during Mmm infection has never been resolved in vivo. Here, we performed lesion-stage-resolved transcriptomic profiling of pathological lung tissue collected from experimentally infected animals and compared to healthy lung tissue collected from unchallenged controls. Differential gene expression and functional enrichment analyses were used to identify biological pathways relevant to Mmm infection and pathological lesion formation. Early infection was dominated by interferon-stimulated genes and cytokine-responsive pathways, creating a primarily antiviral-like response environment despite the bacterial etiology. Red hepatization showed strong induction of neutrophil chemoattractants, epithelial remodeling markers, and early matrix-remodeling enzymes. Consolidation, spanning red and grey stages, was enriched for innate immune activation, leukocyte adhesion, extracellular matrix organization, and persistent interferon signaling. Grey hepatization reflected late-stage consolidation with heightened neutrophil effector activity, oxidative and proteolytic injury, and macrophage and fibroblast-linked collagen processing. Necrosis/Sequestra lesions showed reduced inflammatory signaling, robust extracellular matrix organization, adhesion, and morphogenetic pathways consistent with encapsulation and sequestrum formation. Our data indicate that the dynamic continuum of CBPP lung pathology is initiated by interferon-primed myeloid recruitment and amplified by neutrophil-driven injury and macrophage- and fibroblast-mediated matrix remodeling. These data further substantiate the role of dysregulated immunity in the development of disease during Mmm infection.

Iron is an essential micronutrient that shapes host-pathogen interactions during infection. However, the contribution of iron to the virulence adaptation of the Enterobacter cloacae complex (ECC) remain poorly characterized. This study profiled the effects of iron on E. roggenkampii and E. asburiae clinical isolates. Growth kinetics were assessed in Luria-Bertani broth supplemented with varying iron concentrations and 5% sheep blood, and EDTA. Recovered strains were used for motility and antibiotic susceptibility assays. Phenotypic virulence trait of iron-naive and iron-recovered strains was determined using biofilm formation assays. Whole-genome sequencing was conducted to identify genetic determinants associated with iron acquisition and metabolism. Presence of iron increased bacterial growth, reduced antibiotic susceptibility, and enhanced biofilm formation. At higher iron concentrations, iron-recovered strains exhibited increased biofilm biomass, while there was a high biofilm formation with iron-naive strains at lower iron levels. Genomic analysis identified genes associated with ferrous and ferric iron transport, heme uptake, siderophore biosynthesis, and virulence-related functions, including adhesion and biofilm formation. These findings demonstrate that iron availability and prior exposure modulate ECC physiology and phenotypic traits associated with virulence, supporting a role for iron in shaping adaptive pathogenic potential. Graphical AbstractThe influence of iron metabolism on virulence adaptation of Enterobacter cloacae complex O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737523v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@aa351eorg.highwire.dtl.DTLVardef@855345org.highwire.dtl.DTLVardef@11e0da5org.highwire.dtl.DTLVardef@11f851_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionLomentospora prolificans is a pathogenic filamentous fungus that causes disease primarily in people with severely compromised immune systems. It is pan-resistant to antifungal drugs, but the mechanism of its resistance to amphotericin B (AMB) is unknown. ObjectivesWe aimed to investigate the mechanism of resistance to AMB of L. prolificans. MethodsThe AMB susceptibility of L. prolificans protoplasts was measured using broth microdilution. L. prolificans, either intact, homogenized or fractionated was incubated with AMB in broth. The same activity was carried out with Aspergillus fumigatus as a control. This broth was then used to prepare microdilution plates with Saccharomyces cerevisiae to determine the activity of the conditioned AMB. ResultsAMB was 16-fold more effective in inhibiting the growth of L. prolificans protoplasts than conidia, but only two-fold more effective against A. fumigatus protoplasts than conidia. Incubation of L. prolificans hyphae with AMB in media diminished drug activity to a much greater extent than A. fumigatus, with 8-fold greater fungal mass of the latter required to achieve the effect of the former. Homogenization and fractionization of L. prolificans revealed that the factor inhibiting AMB activity was soluble with a mass >100 kda. DNase, trypsin, proteinase K, amyloglucosidase, SDS and 0.22 m had no effect on the AMB resistance factor, while treatment with urea, acetonitrile inactivated it. ConclusionWe report a different mechanism for AMB resistance based on the existence of a substance residing in the L. prolificans cell wall that can eliminate the antifungal activity of AMB.

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), posing a serious threat to the global swine industry. This study aimed to investigate the effect of CSFV on differential genes of histone lactylation at the H3K18 site in the PI3K-AKT signaling pathway. The site with the most significant change in histone lactylation antibody level was screened by Western blot. Omics analysis was performed using CUT&Tag technology to identify differential genes in the PI3K-AKT pathway between the CSFV-infected group and the mock group, followed by validation using RT-qPCR. Functional analysis of significantly differential proteins was conducted, and the protein expression level of THBS4 was detected by Western blot. The results showed that after CSFV infection of 3D4/21 cells, the H3K18la site exhibited the most significant difference in antibody level. A total of 8,859 differential genes at the H3K18la site were identified by CUT&Tag analysis, including 6,349 up-regulated genes and 2,510 down-regulated genes. Further focusing on the PI3K-AKT signaling pathway, 10 differential genes were identified, comprising 6 up-regulated genes and 4 down-regulated genes. Compared with the control group, the mRNA expression levels of CD19, LAMA1, PDGFRA, BDNF, ANGPT4, and THBS4 were up-regulated in the CSFV-infected group, while FOXO3 and NRTN were down-regulated. Western blot results showed that the protein expression level of THBS4 increased after CSFV infection. These findings lay an important foundation for understanding the molecular mechanisms regulating viral replication and immune evasion, and have significant scientific implications and potential application value.

Many protozoan parasites have complex life cycles entailing migration through different organs in their hosts, but the cues guiding them remain poorly understood. Using a semi-solid plate motility assay, we show that early procyclic forms of Trypanosoma brucei, the first stage to develop in the tsetse fly midgut, perceive several metabolites - including glucose, glycerol and proline - as chemoattractants, while the glycolytic end-product succinate acts as a repellent. During adaptation in the fly, T. brucei switches from glucose/glycerol to proline as its primary energy source. We show that the parasite's chemotactic response towards proline requires adenylate cyclase ACP5 and the cyclic AMP response protein CARP3, two components of signalling pathway involved in pH sensing. These results further support a role for T. brucei's expanded repertoire of receptor adenylate cyclases as environmental sensors that guide navigation through the host.

Neurocysticercosis (NCC), the infection of the central nervous system by Taenia solium larvae, is a leading cause of acquired epilepsy in endemic regions. While viable cysticerci can persist asymptomatically for extended periods, their spontaneous or drug-induced degradation triggers marked perilesional inflammation and severe neurological symptoms. Despite well-documented histopathological characterisation of these lesion states, the host transcriptional programmes associated with viable parasite persistence and early post-treatment lesion disruption remain poorly understood. To address this gap, we performed the first bulk RNA sequencing of pericystic brain tissue using a physiologically relevant porcine model of NCC. Comparing uninfected controls (n = 3), infected untreated pigs with intact viable cysts (n = 6), and antiparasitic-treated pigs with disrupted cysts (n = 3), we identified distinct transcriptional signatures associated with each disease state. Viable infection was associated with broad transcriptional changes (461 upregulated and 175 downregulated genes), characterised by local immune activation alongside suppression of blood-brain barrier (BBB) remodelling, vascular, and neuronal signalling molecular signatures. The post-treatment state with confirmed BBB disruption was associated with a smaller but directionally distinct response (160 upregulated and 57 downregulated genes), marked by inflammatory signalling and increased expression of genes associated with endothelial activation, vascular regulation, and BBB-associated remodelling. Together, these findings suggest that, while immune engagement is a feature shared across both lesion states, the BBB-associated transcriptional axis shifts substantially following treatment. These results provide an exploratory transcriptomic framework for understanding parasite persistence, treatment-induced neuroinflammation, and neurovascular remodelling in NCC, and highlight candidate pathways and genes for future mechanistic investigation.

Tick serine protease inhibitors (serpins) are critical for tick feeding success and pathogen transmission. We previously demonstrated that two unique serpins, S12c5 and S13c5, are tandemly duplicated on Ixodes scapularis chromosome 5 and are injected into the host at heightened levels during feeding by Borrelia burgdorferi (Bb)-infected nymphs. Here, we characterize the biochemical properties and immunomodulatory functions of these serpins and investigate their roles in promoting Bb transmission. Despite their response to Bb infection, S12c5 and S13c5 share less than 20% overall amino acid identity and differ critically at their reactive center loop (RCL) P1 site, which determines substrate specificity: S13c5 contains a basic arginine residue, while S12c5 contains a polar serine residue. Consistent with these differences, recombinant S13c5 efficiently inhibited innate immune proteases including fXa, plasmin, and trypsin IV (SI: 2.5, 1.9, and 1.0; ka: 8.56 x 103, 3.97 x 10, and 1.08 x 10 M-{superscript 1}s-{superscript 1}, respectively), and consequently delayed plasma clotting via the common pathway, whereas rS12c5 did not inhibit these proteases. However, despite molecular modeling predicting stronger interactions between S13c5 and complement proteases, both serpins inhibited MBL-pathway complement activation by targeting MASP1/3. Deletion of the RCL domain abolished this inhibitory activity, confirming both proteins function as typical inhibitory serpins. Functionally, rS13c5 enhanced Bb colonization in C3H mouse organs. Interestingly, immune response assays revealed a trade-off: S13c5, despite its broader inhibitory activity, was less immunogenic than S12c5. Collectively, these findings establish S12c5 and S13c5 as Bb transmission factors that promote spirochete survival through disruption of host innate immunity.

GlnH is an amino acid binding protein that senses aspartate to regulate metabolism via the PknG pathway in diverse Actinobacteria. Information about ligand occupancy of periplasmic GlnH is conveyed to PknG via an uncharacterised transmembrane protein GlnX. This pathway is important in the virulence of Mycobacterium tuberculosis, and in regulating valuable industrial fermentations by Corynebacterium glutamicum. GlnH has a "Venus flytrap"-like structure, comprising two lobes that surround the ligand aspartate. However, the conformational changes that allow GlnH to initiate this signalling pathway are unknown. To address this question, we produced GlnH from pathogens M. tuberculosis and Mycobacterium marinum and non-pathogens Mycobacterium smegmatis and C. glutamicum and used X-ray crystallography and cryo-EM to determine their structures. The results show that amino acid specificity is conserved in all homologues. However, GlnH from Mycobacteria was monomeric and bound aspartate with nanomolar affinity, whereas GlnH from C. glutamicum bound aspartate with micromolar affinity and dimerised upon binding. Whilst GlnH of the non-pathogens was stable at neutral pH, GlnH from the pathogens was most stable at acidic pH, reflecting the environment of host phagosomes. Structures were determined for all homologues, but only M. smegmatis GlnH crystallised in both unbound (Apo) and Asp-bound forms. GlnH has an open structure with a cleft between the lobes to permit access to aspartate. The Asp-bound structure is more compact with the lobes locked together, completely enclosing the ligand. AlphaFold was used to design mutations to disrupt the predicted GlnH-GlnX interface, and these variants failed to complement the metabolic defect of glnX knockout in M. smegmatis, supporting the predicted complex and suggesting how the GlnH conformational change is transmitted GlnX to initiate signalling.

Drug-resistant tuberculosis and the rising incidence of nontuberculous mycobacterial (NTM) infections are a growing concern that demands innovative therapeutic strategies. Despite advances in diagnostics, drug discovery, and vaccine strategies, significant gaps remain. Mycobacteriophages and their lytic enzymes offer a promising solution due to their natural abundance and diversity, host specificity and ability to disrupt complex cell envelopes and biofilms. In this study, we report the genomic and functional characterization of a V-Cluster mycobacteriophage, EniyanLRS, isolated near a hospital in Delhi and the encoded endolysins LysA and LysB. EniyanLRS features a 78.53 kbp genome with a notably low GC content (56.9%) as compared to other mycobacteriophages, and an exceptionally long Tape Measuring Protein (TMP) gene (5.97 kbp). Its genome lacks genes related to lysogeny and harbours 24 tRNAs, suggesting high translational efficiency. Phenotypically, EniyanLRS exhibits a siphovirus morphology, lytic lifecycle and infects Mycobacterium smegmatis and drug-resistant Mycobacterium fortuitum. LysA, with its lysozyme-chitinase-amidase domain architecture, did not demonstrate significant antibacterial or antibiofilm activity. Conversely, LysB, an /{beta}-hydrolase, exhibited superior in vitro esterase activity compared to previously reported LysB enzymes and showed pronounced cell wall disruption of M. smegmatis and M. fortuitum, along with considerable antibiofilm efficacy (62.77% and 41.91% inhibition, respectively). Collectively, these findings highlight the potential of EniyanLRS and its LysB enzyme as potent biocontrol agents against pathogenic mycobacteria, which can be explored to treat planktonic cells and biofilm-associated infections.