Virulence

Cell-mediated immune responses are crucial for protecting the host against Toxoplasma gondii infection. However, impaired immunity, such as T-cell exhaustion, is a common phenomenon during chronic infection. This may represent a strategy employed by T. gondii to evade host defenses. T-cell immunoglobulin and mucin-domain containing 3 (Tim-3) is an important regulatory molecule involved in cell-mediated immunity. This study examined the expression of Tim-3 and the effects of its blockade in a mouse model of toxoplasmosis. In mice with chronic T. gondii infection, we found that Tim-3 is highly expressed in both cyst-bearing and non-cyst-bearing tissues, and its expression correlates with the parasite burden. Blocking the Tim-3 pathway with an anti-Tim-3 antibody enhances the immune response, resulting in elevated levels of cytokines (IFN-{gamma}, IL-12p70, IL-2, IL-9) and the chemokine CXCL1 in the serum, increased leukocyte infiltration (CD3+, CD14+ cells) in the brain, and downregulation of Tim-3 expression in microglial cells. As a result, the anti-Tim-3 treatment resulted in a 62% reduction in the number of tissue cysts and a trend towards an increase in the homeostatic signature, P2RY12, in microglia. Our study provides proof of concept for an anti-Tim-3 approach in treating chronic T. gondii infection and potentially other brain-residing pathogens.

The tobacco hornworm moth (Manduca sexta) is evaluated as a model of bacterial virulence and host-pathogen dynamics. Infections of Pseudomonas aeruginosa were established by injection of 5th-instar larvae, and multiple assays of virulence were evaluated. Infected larvae exhibited dose-dependent mortality, reduced growth, melanization, behavioral changes, and altered frass constitution. Even low-dose infections that were not fatal exhibited impaired growth, but individual growth trajectories revealed considerable heterogeneity among worms given the same dose. Twice-daily antibiotic treatment with gentamicin or cefepime improved survival four- to five-fold but did not rescue 100%. Heat-killed cells and filtered culture supernatant alone induced significant morbidity and mortality, suggesting secreted bacterial products are important to pathogenesis. Bacterial burden analysis revealed a shifting bacterial distribution over time, with decreasing hemolymph titers and increasing localization in fat body, gut, and carcass. Hornworms thus offer a more sensitive analysis of bacterial infection dynamics and consequences than do larvae of the more commonly used wax moth.

Treponema pallidum ssp. pallidum, the causative agent of syphilis, has a small proteome and encompasses numerous strains. Knowledge gaps remain in understanding the molecular mechanisms of pathogenesis of this bacterium, as well as the structure and function of the full complement of proteins encoded by T. pallidum. Here, an AI-based structure-to-function modeling workflow was used to investigate the complement of proteins encoded by T. pallidum. High-confidence structure models were generated for 976 T. pallidum proteins, covering 99% of the proteome. Analysis of the generated models using the protein structure comparison server DALI enabled high-confidence, structure-based functional annotation of 877 T. pallidum proteins, including 240 of the 323 proteins of unknown function encoded by this pathogen. Additionally, 63 putative pathogenesis related proteins (PPRPs) and seven treponemal proteins with previously uncharacterized similarity to outer membrane proteins (OMPs) from Gram-negative bacteria were identified. A workflow for B cell epitope (BCE) prediction identified 1133 surface-exposed, host-facing potential epitopes in known and predicted T. pallidum OMPs, of which 92 were prioritized based on bioinformatic analyses, biophysical properties, amino acid sequence conservation, and previous protein expression data. This work provides insight into T. pallidum pathogenesis through structure modeling-based functional annotation, including characterization of proteins of unknown function. This study also informs syphilis vaccine design by identifying new potential T. pallidum OMPs, as well as host-facing regions of T. pallidum OMPs that have conserved amino acid sequences in globally circulating strains. Statement of importance/impactThis study presents the first AI-based global structure modeling-to-function analysis of the proteome of Treponema pallidum, the bacterium that causes syphilis. Structure-based functional predictions of previously uncharacterized proteins, including proteins potentially involved in virulence, provide novel insight into mechanisms of pathogenesis. The work also informs syphilis vaccine development by the identification and structural characterization of new candidate vaccine proteins in globally circulating strains of T. pallidum.

Ehrlichia ruminantium, the causative agent of heartwater disease, is an obligate intracellular bacterium that poses significant economic threats to livestock production in endemic regions. Current research models present substantial ethical, logistical, and economic constraints, particularly for studying host-pathogen interactions within arthropod vectors. Here we establish Galleria mellonella larvae as a tractable invertebrate infection model for Ehrlichia ruminantium, enabling experimental investigation of pathogen persistence and host-pathogen interactions in an arthropod system. Following infection, G. mellonella proved susceptible to E. ruminantium with moderate mortality and remarkable bacterial persistence. Using rhodamine-labeled bacteria and fluorescence microscopy, we tracked bacterial dissemination from injection sites to systemic distribution in characteristic segmental patterns throughout the larval body. Critically, we confirmed intracellular localization of E. ruminantium within hemocytes, the primary immune cells of G. mellonella. Quantitative PCR analysis revealed stable bacterial loads over the study period, indicating bacterial persistence within the host. These findings demonstrate that E. ruminantium can hijack the innate immune system of G. mellonella, similar to its behavior in natural hosts. The segmental bacterial distribution suggests exploitation of hemolymph circulation and sessile hemocyte populations, providing new insights into potential mechanisms of pathogen persistence. This model offers significant advantages: ethical acceptability, cost-effectiveness, experimental tractability, and compatibility with high-throughput screening approaches. The G. mellonella system represents a valuable complement to existing mammalian models and provides a unique platform for investigating arthropod-specific aspects of E. ruminantium biology, screening antimicrobial compounds, and understanding mechanisms of immune evasion that may inform strategies for heartwater disease control.

Bilophila wadsworthia is a gut pathobiont implicated in dysbiosis-driven inflammation, yet its pathogenic mechanisms remain poorly investigated. Here, we evaluated the suitability of Galleria mellonella larvae as an in vivo model to study B. wadsworthia infection. Two infection routes were compared: oral inoculation to mimic gastrointestinal colonization and hemolymph injection to model systemic infection. Oral challenge had minimal impact on larval health, whereas hemolymph injection caused marked morbidity, including reduced mobility, impaired cocoon formation, and progressive melanization, indicating that access to the circulatory system is required for overt disease. Infection required live bacteria, with B. wadsworthia capable of intracellular replication within hemocytes, leading to transient depletion of circulating immune cells followed by compensatory hemocyte proliferation. These findings reveal tight coupling between bacterial proliferation and host immune dynamics. Comparison with other sulfidogenic bacteria suggests that Bilophila pathogenicity is likely to involve host-specific interactions. Overall, our results establish G. mellonella as a practical and ethically favorable model to investigate B. wadsworthia virulence, host-pathogen interactions, and mechanisms relevant to gut-associated infection.

Mycoplasma synoviae is an avian pathogen that causes respiratory disease and synovitis, and its hemagglutinin plays a critical role in host cell adhesion. However, the key residues and structural mechanisms underlying hemagglutination remain unclear. In this study, domain analysis of the hemagglutinin family of Mycoplasma synoviae revealed that it contains long-chain and short-chain types, among which LAM HA (VY93_RS01465) was selected as the bait protein due to its complete C-terminal conserved domain. Through yeast two-hybrid screening, 18 host proteins interacting with LAM HA were identified. Furthermore, five key amino acid residues S83, R85, Y88, N124, and K192 were found to mediate hemagglutination activity. Deletion of these residues reduced the hemagglutination titer of LAM HA under acidic conditions. Secondary structure analysis showed that the deletion mutation decreased the -helix content while increasing the proportions of {beta}-sheet and random coil. Molecular dynamics simulations revealed that the mutant exhibited generally higher root mean square deviation and root mean square fluctuation values than the wild-type under different pH conditions, with a marked decrease in structural stability particularly at pH 5.0 and 6.0. These findings indicate that LAM HA, as a critical adhesin, exerts its hemagglutination function dependent on specific key residues and pH-sensitive conformational stability. IMPORTANCEMycoplasma synoviae (M. synoviae) causes significant economic losses to the poultry industry worldwide. Lipid-related membrane protein hemagglutinin (LAM HA) is a surface adhesin essential for host cell attachment, but its precise amino acid residues and structural features have not been defined. In this study, five key residues (S83, R85, Y88, N124, and K192) were identified as critical for LAM HA-mediated hemagglutination activity. Deletion of these residues altered the secondary structure composition, reduced conformational stability under acidic pH conditions, and decreased hemagglutination activity. These findings reveal a previously unknown structure-function relationship of M. synoviae LAM HA, demonstrating that its hemagglutination activity depends on specific residues and pH-sensitive structural integrity. This provides new insights into the molecular mechanisms of M. synoviae adhesion and offers potential targets for the development of novel intervention strategies against avian mycoplasmosis.

Secreted virulence factors (e.g., hydrolytic enzymes, toxins, agglutinins) play an important role in human diseases. Nevertheless, their secretion by some pathogenic fungi, especially some virulent Candida-related species such as Candidozyma auris, is still only partly characterized. Here we used high-throughput mass-spectroscopy analysis to identify polypeptides secreted by C. auris into growth medium under two physiologically relevant pH conditions: pH 5.5 and pH 7.5. This analysis revealed that many secreted polypeptides belong to putative virulence factors and enzymes involved in cell wall biogenesis. Moreover, we found that 13 and 27 polypeptides were detected only at pH 5.5 or pH 7.5, respectively. Furthermore, our findings indicate that lower pH (pH 5.5) favours secretion of several putative virulence factors including aspartic proteases and polypeptides potentially facilitating host-pathogen interactions. In contrast, the majority of polypeptides detected only at pH 7.5 are involved in N-glycosylation and protein folding. Thus, this secretome analysis reveals numerous C. auris polypeptides with putative roles in infection and host-pathogen interactions. Moreover, their differential secretion at pH 5.5 and pH 7.5 may reflect different strategies used by C. auris to elicit infections in different anatomical sites.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

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.

The black legged tick, Ixodes scapularis, is a vector of the bacterium that causes Lyme disease and several other illnesses, including anaplasmosis, babesiosis, and tick-borne encephalitis. Although high-quality genome annotations are available for I. scapularis, functional understanding of I. scapularis genes is limited. To address this, we developed a platform for genome-wide CRISPR-Cas9 knockout screening in I. scapularis cells. To evaluate the platform, we performed a screen to identify genes associated with cellular fitness, and screens for resistance to treatment with copper chloride, Antimycin A, or Destruxin A (DA), a cyclic hexadepsipeptide produced by the pathogenic fungus Metarhizium anisopliae. In each case, the screens implicate specific sets of conserved and non-conserved I. scapularis genes in relevant cellular functions, providing the first experimental evidence of function for a large set of I. scapularis genes. Altogether, in this first-of-its-kind effort for the arthropod subclass Acari, we present an unbiased genome-wide CRISPR-Cas9 knockout cell screening platform, related resources, and datasets that will be broadly useful to efficiently uncover cellular functions of I. scapularis genes.

AbstractEmergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterisation of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of H. capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences, to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall (1,3)-glucan by targeting the transcripts of the -glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus. ImportanceEmergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognised in recent years and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (1) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (2) methods for knocking down gene expression in E. africanus, and (3) the permanent disruption of genes through CRISPR/Cas9 gene editing.

Parasitic flatworms, including cestodes and trematodes, are covered by a specialized syncytial tegument that mediates nutrient uptake and host-parasite interactions. While the tegument of trematodes has been extensively characterized, its molecular composition in cestodes remains largely unknown. In this work, we performed a comparative proteomic analysis of the tegument of three cestode species, including larval and adult stages: Hymenolepis microstoma, Mesocestoides corti (syn. M. vogae) and Echinococcus multilocularis. Using stringent enrichment criteria relative to whole-worm extracts, we identified hundreds of tegument-enriched proteins in each species. Comparative analyses revealed a conserved core of tegumental proteins shared among all three species, including members of the Tegument Allergen-Like (TAL) family, vesicular trafficking components and calcium-sensing proteins, and identified candidates for nutrient uptake activities such as glucose and nucleoside transporters. Further comparative analyses revealed a set of shared tegumental proteins with the trematode Schistosoma mansoni, including conserved proteins that are specific to parasitic flatworms, supporting the existence of a conserved ancestral tegumental proteome. Finally, we confirmed tegumental expression of several candidate genes in H. microstoma and E. multilocularis, and demonstrated regionally restricted gene expression among tegumental cytons, suggesting functional specialization within the syncytial tegument. Altogether, these results reveal an evolutionarily conserved composition of the tegument of parasitic flatworms, providing a foundation for future work targeting this critical host-parasite interface.

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium that causes scrub typhus, a potentially life-threatening disease. To systematically identify host factors regulating early stages of infection, we performed a microscopy-based genome-wide siRNA screen in HeLa cells. This approach identified 2,989 genes grouped into 55 functional networks that modulate bacterial entry and intracellular translocation. In addition to confirming previously described pathways, including endocytosis and microtubule-dependent trafficking, the screen revealed an association between Ot infection and host cell cycle regulation. We found that Ot preferentially infects and/or replicates in host cells in the S and G2 phases, where intracellular bacterial accumulation is increased relative to G1. Early infection was associated with a shift in host cell cycle distribution, consistent with a delay in progression through S and G2 phases. Longitudinal analysis further showed that these cell cycle states support enhanced bacterial expansion. In parallel, infected cells exhibited reduced proliferation compared to uninfected cells, suggesting that Ot infection alters host cell division dynamics. Together, these findings support a model in which host cell cycle state influences susceptibility to Ot infection and intracellular growth. This work provides a systems-level map of host pathways involved in early infection and identifies cell cycle regulation as an important component of host-pathogen interactions in scrub typhus. Author SummaryScrub typhus is a potentially life-threatening disease caused by the bacterium Orientia tsutsugamushi, which can only survive and replicate inside human cells. Although some host factors involved in infection have been identified, many remain unknown. In this study, we used a large-scale screening approach to systematically identify human genes that influence the bacteriums ability to enter and move within host cells. Our analysis uncovered multiple pathways required for infection, including a role for the host cell cycle. We found that O. tsutsugamushi preferentially accumulates in cells during specific stages of the cell cycle, particularly when cells are preparing to divide. At the same time, infection slows host cell division, suggesting that the bacterium alters the cellular environment to support its own growth. These findings provide new insight into how O. tsutsugamushi interacts with human cells and identify potential host processes that could be targeted to limit infection.

Orientia tsutsugamushi (Ot) is an obligatory intracellular bacterium that can cause scrub typhus, an emerging but severely neglected disease with high mortality rates. Ot Karp and Gilliam strains account for most reported cases in Southeast Asia. Our group has reported that Karp-infected outbred and inbred mice exhibit more severe disease outcomes than their Gilliam-infected counterparts, likely due to their excessive inflammation and tissue injury. Macrophages (M{Phi}s) serve as the main target cells for Ot replication and host defense against the infection; they also are key players in immune modulation and cytokine/chemokine production. However, it remains unclear as to how M{Phi}-Ot interactions impact disease outcomes. In this study, we focused on RNAseq from C57BL/6 mouse-derived M{Phi}s to reveal Ot infection- and strain-related immune signatures. While Ot infection modulated several common canonical pathways/genes, we identified unique pathway/gene signatures that were highly selective for Karp or Gilliam strain, respectively. Karp infection uniquely upregulated proinflammatory signaling and pattern recognition receptors, including C-type lectin receptors (CLRs), including Mincle/Clec4e and Dectin-2/Clec4n. In contrast, Gilliam infection enhanced M{Phi} proliferation and DNA replication, and Gilliam strain grew better in M0-like M{Phi}s than Karp strain. In IFN-primed (M1-like) M{Phi}s, however, Karp exhibited significantly greater resistance against host killing than Gilliam, suggesting its superior ability to evade host immune responses. Overall, Karp strain preferentially upregulated CLRs and activated type 1-skewed inflammatory responses, but it is also relatively resistant to IFN-mediated killing. This study provides new insights into potential mechanisms underlying Ot strain-associated immune responses and disease outcomes. Author SummaryOrientia tsutsugamushi (Ot), the causative agent of scrub typhus, is an understudied, life-threatening pathogen endemic to Southeast Asia. Among its various pathogenic strains, Karp and Gilliam are clinically dominant and exhibit distinct disease severity. Building on previous reports that Karp-infected mice display more severe disease than those infected with Gilliam, we explored how strain-specific interactions with macrophages (M{Phi}s), a central cell type in both Ot replication and host immunity, may contribute to these outcomes. Using RNAseq of primary M{Phi}s from C57BL/6 mice, we characterized transcriptional profiles of both strains. Karp infection was uniquely associated with increased proinflammatory signaling and the selective induction of bacterial-sensing molecules. In contrast, Gilliam infection promoted host cell proliferation and supported more efficient bacterial replication in naive M{Phi}s. Notably, Karp demonstrated increased resistance to host killing in stimulated (proinflammatory) M{Phi}s compared to Gilliam. These findings suggest that the Karp strain can drive strong inflammatory responses but is adept at evading IFN-mediated immune defenses. Our study uncovers key immunological distinctions between Ot strains and offers novel insights into the mechanisms of strain-specific pathogenesis in scrub typhus, with potential to uncover biomarkers for disease severity.

Whipworms (Trichuris spp.) are intracellular intestinal parasites that develop within the host caecal epithelium, yet the host signals that regulate their growth and developmental progression remain poorly understood. Progress in studying these processes has been limited by the lack of physiologically relevant in vitro systems capable of supporting sustained whipworm development. Here, we established an in vitro infection system using caecal organoids (caecaloids) and evaluated their capacity to support sustained growth and morphological development of Trichuris muris larvae. To rigorously validate this system, we generated a comprehensive and up-to-date anatomical and biometrical reference dataset describing the whole-body growth and tissue-level morphogenesis of T. muris throughout its life cycle in vivo. Quantitative analysis across larval and adult stages confirmed that the trajectory of parasite growth is largely conserved across host mouse strains and provided a detailed contextualised description of the development of key anatomical structures of T. muris. Using this reference framework, we evaluated parasite growth and development in long-term T. muris-caecaloid co-cultures. Larvae invading the caecaloid epithelium remained intracellular within syncytial tunnels and exhibited sustained growth over extended culture periods. in vitro parasites developed increasing anatomical complexity, including formation of the bacillary band, stichosome, intestine, and rectum. Importantly, quantitative comparisons revealed that larvae developing within caecaloids follow growth trajectories and morphological developmental patterns closely resembling those observed in vivo. This study therefore presents the first detailed anatomical and morphometric framework for validating whipworm development in an organoid system and provides concrete evidence that the caecaloid epithelium is sufficient to trigger and sustain whipworm growth and morphogenesis, establishing caecaloids as a powerful experimental platform for investigating Trichuris infection and development.

Microtubules (MTs) play central roles in the organization and morphology of trypanosomatid parasites, forming highly specialized cytoskeletal structures such as the subpellicular corset, the flagellar axoneme, and the mitotic spindle. Functional specialization of MTs is regulated by the "tubulin code", which is defined by the combination of different - and {beta}-tubulin isotypes, a set of post-translational modifications (PTMs) and specific MT-binding proteins. Although multiple tubulin PTMs have been described in trypanosomatids using specific antibodies or mass spectrometry, to date no comprehensive mapping has been reported in Trypanosoma cruzi, the causative agent of Chagas Disease. In the present work, we performed a high-resolution proteomic analysis of PTMs present in - and {beta}-tubulin subunits of the T. cruzi Dm28c strain, using tubulin-enriched extracts obtained by in vitro polymerization. Multiple PTMs were identified, including acetylation, methylation, phosphorylation, and polyglutamylation, for which many modified amino acids had not been previously reported in trypanosomatids. Structural mapping of these modifications onto a predicted /{beta}-tubulin heterodimer showed that most modified residues are located in solvent-exposed regions of the protein. Together, these findings provide the first systematic map of tubulin PTMs in T. cruzi and support the existence of a complex tubulin code contributing to microtubule regulation in this parasite.

Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites. IMPORTANCETrypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.

Candida auris is an emerging fungal pathogen posing a serious global health threat due to its high transmissibility and multidrug resistance profile. Despite recent molecular advances in scrutinizing this enigmatic microbe, much of our understanding in regards to its pathomechanisms still remain unelucidated. Since, microbial pathogenesis is modulated by a dynamic interplay between the host and the pathogen, dissecting such host-pathogen interaction involving C. auris can shed novel insights into its pathogenic cascade. As such, to further characterize the virulence repertoire of C. auris, this study applied an integrated quantitative proteomics strategy to scrutinize early-phase of infection. We utilized an in-vitro and an in-vivo experimental setup based on immune cells and murine model. Integrated proteomic analysis revealed a coordinated remodelling of cellular processes by C. auris during host-pathogen interaction, including downregulation of translational machinery, and modulation of molecules involved in metabolic rewiring, stress-response, and structural rearrangements. Collectively, these findings suggests that survival of C. auris under host-immune pressure is accompanied by rapid context-dependent molecular adaptations. IO_SCPLOWMPORTANCEC_SCPLOWCandida auris is a critical high priority fungal pathogen classified by the World Health Organization (WHO) that constitute a serious threat to global health. Often termed as a superbug due to its high transmissibility and multidrug resistant profile, the microbe has spread across the globe and is capable of causing high mortality rates. Molecular studies scrutinizing the pathogenic mechanisms of C. auris are limited and represents a major bottleneck to decipher and device intervention strategies against this enigmatic pathogen. As such, this study is aimed at widening the molecular knowledge spectrum of C. auris in regards to its virulence and pathogenesis. Here we dissect the host-pathogen interaction of C. auris by establishing experimental infection models and subsequently applying an integrated proteomics strategy to capture the organisms virulence repertoire modulating fungal pathogenesis.