mSphere

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

PurposeElucidate the influence of glucose metabolic pathways on A. fumigatus lung and corneal infection. MethodsThe A. fumigatus acuF and creA genes were deleted in an mcherry-expressing strain. The mutants were tested for alterations in radial growth, cell wall composition by fluorescence staining assays, and antifungal sensitivity through broth microdilution assays. Hyphal penetration of the strains through explanted porcine corneas was tracked by confocal microscopy using the mCherry signal. Virulence was evaluated in established models of invasive pulmonary aspergillosis (IPA) and fungal keratitis (FK) using C57BL/6J mice. ResultsDeletion of the A. fumigatus phosphoenolpyruvate carboxykinase (acuF) resulted in a dependency on exogenous glucose to support growth in vitro, but did not impact virulence in either the IPA or FK models. Loss of the carbon catabolite repressor CreA resulted in a broad dysregulation of carbon metabolic pathways and altered cell wall homeostasis. Surprisingly, whereas the{Delta} creA remained fully virulent in the lung, the mutant was unable to establish infection in the FK model. This in vivo phenotype corresponded to an inability of{Delta} creA to physically invade porcine corneal explants, which we attributed to a marked reduction in cell wall chitin content. ConclusionsGluconeogenesis is dispensable for A. fumigatus lung and corneal infection, suggesting tissue-derived glucose supports fungal growth in both environments. Loss of CreA disrupts glucose assimilation, its synthesis into chitin and, consequently, cell rigidity and hyphal invasion into the dense corneal stroma. Thus, CreA and other cell wall regulatory proteins may serve as targets for novel FK antifungals.

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised patients. Alveolar macrophages are the first line of defense against Cryptococcus infection. Our previous study showed that deletion of Cdc50, the regulatory subunit of P4-ATPase (lipid flippase) complex, results in increased phagocytosis and macrophage killing, and avirulence in animal models. However, how fungal flippase dysfunction modulates Cryptococcus-macrophage interaction remains unknown. Here we identify Cdc50 as a central determinant of membrane lipid homeostasis, extracellular vesicle (EV) biogenesis and macrophage responses in C. neoformans. Our whole cell lipidomic analysis revealed that loss of Cdc50 disrupted membrane lipid homeostasis leading to phospholipid enrichment in cdc50{Delta} mutant, and a reduction in fatty acid production accompanied by pronounced ultrastructural defects in membrane architecture. Loss of Cdc50 also induced a hyper-vesiculating phenotype, with cdc50{Delta} producing significantly more extracellular vesicles (EVs) than wild type H99 cells. Lipidomic profiling of cdc50{Delta} EVs revealed enrichment of phospholipids, including phosphatidylserine (PS), indicating active lipid sorting during vesicle biogenesis. Functional analysis showed that EVs from the wildtype H99 suppress phagocytosis whereas cdc50{Delta} EVs enhance phagocytosis, indicating a differential macrophage priming. Despite increased PS externalization in cdc50{Delta} cells and EVs, macrophage recognition and uptake occur independent of PS-mediated efferocytosis pathways, including PS receptor MertK. Following macrophage uptake, cdc50{Delta} were intrinsically vulnerable to macrophage killing due to rapid phagosome acidification. Together, we demonstrate that Cdc50 dependent lipid homeostasis regulates EV production, lipid composition, membrane architecture and drives the intracellular fate of C. neoformans. IMPORTANCECryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised individuals. Understanding how this pathogen evades host immune mediated clearance is essential for developing new treatment strategies. Here, we demonstrated that Cdc50, the regulatory subunit of fungal lipid flippase complex, regulates membrane lipid homeostasis that governs extracellular vesicles (EV) biogenesis and macrophage immune responses. Loss of Cdc50 drives global membrane lipid remodeling, hyper-production of phospholipid enriched EVs that enhance macrophage phagocytosis, while the wild-type EV reduce macrophage phagocytosis. Contrary to the prevailing assumption that phosphatidylserine (PS) externalization on the fungal surfaces mimics the mammalian "eat-me signal", we show fungal PS does not engage canonical PS receptor MertK, revealing a fundamental difference between fungal and mammalian PS biology. Furthermore, cdc50{Delta} cells are unable to resist phagosomal acidification, rendering them susceptible to macrophage killing. These findings establish how phospholipid homeostasis contributes to early host-pathogen interactions and serves as a compelling antifungal target in cryptococcosis.

Daptomycin resistance (DAP-R) in enterococci is associated with alterations in the membrane lipid composition. The membrane-bound protein MprF is responsible for the synthesis of amino acid-modified lipids in bacteria, and these modified lipids contribute to DAP-R in some Gram-positive pathogens. In enterococci, MprF synthesizes three lysine-modified lipids: the phospholipid lysyl-phosphatidylglycerol (Lys-PG), and the newly identified cationic glycolipids lysyl-diglucosyl-diacylglycerol (Lys-Glc2-DAG) and lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG). Given the recent discovery of cationic glycolipids in enterococci, we re-examined a collection of laboratory-evolved DAP-R E. faecalis to investigate whether these lipids contribute to DAP-R. We found that levels of Lys-Glc2-DAG were strikingly reduced in DAP-R variants with high-level resistance. The dramatic alterations in Lys-Glc2-DAG levels were temporally coupled with the emergence of loss-of-function mutations in the gene drmA, which encodes a DUF998 family protein of unknown function. DrmA is a membrane protein with six predicted transmembrane helices and is widely distributed among Gram-positive and Gram-negative bacteria, including plant and animal pathogens. Complementation of the DAP-R strains with wild-type E. faecalis drmA significantly lowered their DAP MIC, reversing their trajectory to high-level DAP-R. Using genetic and lipidomic approaches in the natively DAP-sensitive strain OG1RF, we conclusively linked drmA loss-of-function with significantly reduced Lys-Glc2-DAG levels as well as a small but significant increase in Lys-PG levels. Yet, drmA inactivation in OG1RF did not alter its DAP MIC. We conclude that drmA loss-of-function confers elevated DAP MIC on the background of preceding mutations in the DAP-R evolutionary trajectory, most likely mutations in cls1. The recurrence of drmA mutations in multiple studies underscores its importance in DAP-R evolution. Overall, our work identifies a role for the DUF998 family in cellular lipid homeostasis and confirms its significant role in the evolution of DAP-R.

Cryptococcus neoformans and related species are major human pathogens that cause cryptococcosis, a disease with high mortality and morbidity despite antifungal therapy. Pathogenic Cryptococcus spp. cells express a polysaccharide capsule, which is the most important virulence factor. In this study we analyzed the distribution of capsule sizes for several strains from Cryptococcus spp. and found that they follow stochastic dynamics, with a heavy right-hand tail distribution, favoring larger capsules. The distribution for each strain is remarkably stable despite repeated perturbation of culture conditions including media refreshment, time, and macrophage ingestion. Growth in macrophages resulted in different capsule distributions, observed in vitro, with a suggestion of different polysaccharide-like materials formed or utilized in the resident phagosome. We propose that the stability in capsule size distributions represents a capsulestat mechanism for the population. An emergent property whereby individual cells manifest capsule size variation emanating from random effects on individual capsule assembly steps. This distribution balances between cells with large capsules that are less susceptible to a variety of environmental stresses at the price of slower replication, increased size, and increased energy requirements and cells with smaller, less protective capsules that reproduce faster. Thus, Cryptococcus spp. populations establish a bet hedging strategy that can enhance the viability of the population as conditions change at the cost of optimal short-term growth.

The eukaryotic pathogen Leishmania major causes disfiguring cutaneous lesions, whose resolution can be complicated by secondary bacterial infections. Bacteria, including Aeromonas spp., also interact with L. major promastigotes in the sandfly midgut. The mechanisms by which L. major competes with bacteria and resists their toxins are poorly defined. Prior work proposed that L. major resists the Aeromonas-produced pore-forming toxin aerolysin using an altered GPI-anchor. However, we found that L. major is sensitive to aerolysin. Here, we determined the mechanism by which L. major promastigotes are sensitive to aerolysin, using flow cytometry and biochemical approaches to analyze promastigotes genetically deficient in enzymes that produce key membrane components. The virulence factor lipophosphoglycan protected L. major from aerolysin cytotoxicity. The metalloproteinase GP63 exerted the necessary furin-like protease activity to activate aerolysin. Leishmanial GPI-anchored proteins were necessary for aerolysin heptamerization and killing of L. major promastigotes. Finally, mutation of the GPI-anchor binding domain of aerolysin crippled its cytotoxicity, consistent with its reliance on the GPI-anchor binding site to engage GPI anchors on the surface of L. major promastigotes. Taken together, we propose the L. major virulence factor lipophosphoglycan defends against pore-forming toxins made by bacterial competitors, while the GP63 metalloproteinase activates pro-aerolysin like furin. Overall, this study highlights approaches microbes use to compete with each other. Graphical AbstractAerolysin cytotoxicity depends on gp63 and LPG in Leishmania major promastigotes. (A) Wild type Leishmania major promastigotes are sensitive to aerolysin, which forms lethal heptameric pore complexes in the plasma membrane (B) L. major lpg1-- promastigotes are highly sensitive to aerolysin challenge because they lack LPG. (C) L. major gp63-- promastigotes have wild type sensitivity to aerolysin challenge but resist pro-aerolysin. (D) L. major gpi8-- knockout promastigotes are resistant to aerolysin and show no heptameric pore complexes in the plasma membrane. Created in BioRender.

Fungal infections are an escalating global health concern, with rare Candida species posing an urgent threat due to emerging multidrug resistance. Clavispora (Candida) lusitaniae is an uncommon pathogen in which multidrug resistance has been documented during antifungal therapy, yet the selective forces driving this phenotype remain unclear. Here, we show that exposure to the echinocandin micafungin (MCF) alone can select for multidrug resistance in C. lusitaniae. Through controlled evolution experiments we identified individual point mutations in genes encoding ergosterol biosynthesis enzymes (ERGs), sterol trafficking proteins (OSH2), and the echinocandin drug target (FKS1) that confer a significant fitness benefit to one or more classes of antifungals. We find that ERG loss-of-function is the primary and independent driver of pan-antifungal resistance to echinocandins, azoles and polyenes. The ERG mutants have <1% ergosterol, increased levels of non-toxic sterol intermediates, and increased chitin content, consistent with both cell membrane and cell wall remodeling that enables the fungal pathogen to evade all three drug classes. The convergence of sterol reprogramming and compensatory cell wall remodeling that occurs during adaptation to echinocandin monotherapy can evolve through a single point mutation and parallels our recent case study of acquired multidrug resistance.

Pathogens colonise metabolically diverse host environments. How metabolites found in host environments regulate antimicrobial drug susceptibility remains to be fully understood. Here we report on the roles of gut metabolites, short chain fatty acids (SCFAs), in antifungal drug susceptibility of the gut commensal and fungal pathogen Candida albicans. A genetic screen revealed that C. albicans mutants in peroxisome biogenesis display increased tolerance to the antifungal drug fluconazole. Peroxisomes are important for the metabolism of SCFAs by beta-oxidation, and exposure to the SCFAs butyrate and crotonate increased susceptibility and reduced tolerance to fluconazole. To understand if SCFAs inhibit fluconazole tolerance through their ability to inhibit histone deacetylases (HDACs), we compared them with the HDAC inhibitor trichostatin A. These experiments did not reveal an obvious connection between the degree of HDAC inhibition and the degree of fluconazole tolerance reduction. Exposure of C. albicans to crotonate and butyrate revealed transcriptional reprogramming involving remodelling of acetyl-CoA metabolism by upregulation of genes for beta-oxidation, peroxisome biogenesis and intracellular transport of acetyl-CoA, while the expression of ergosterol biosynthesis genes was reduced. Since ergosterol gene expression is required to overcome fluconazole stress, these results explain how SCFAs reduce fluconazole tolerance. Taken together, our results implicate peroxisome biogenesis and metabolism in fluconazole susceptibility. We posit that balanced acetyl-CoA metabolism promotes sufficient ergosterol biosynthesis to overcome fluconazole stress and drive tolerant growth. These pathways are perturbed by metabolic changes induced by SCFAs. These findings add to our understanding of the importance of metabolic regulation in antimicrobial drug responses.

Fungi play important roles in health and disease, but current methods such as culture, PCR, and amplicon sequencing cannot provide genome-level characterization directly from clinical samples. Although metagenomic sequencing could overcome these limitations, it remains impractical in clinical samples where fungal DNA is present at low abundance relative to human DNA. Here, we extend a recently described microbial enrichment method (MEM)(1) to fungi (fungal Microbial Enrichment Method; fMEM) and test the method in bronchoalveolar lavage (BAL) samples to demonstrate direct-from-sample fungal metagenomic analysis and metagenome-assembled genome (MAG) recovery. In BAL samples, fMEM depleted human DNA by more than 1000-fold while preserving fungal DNA within 10-fold, enabling shotgun sequencing from samples with fungal biomass as low as 10 pg fungal DNA per 200 {micro}L BAL. fMEM enabled de novo recovery of fungal MAGs from three of four sequenced BAL samples, including two near-complete MAGs (>90% BUSCO completeness) and one 82.1% complete MAG, with low BUSCO-estimated contamination ([&le;]1.5%). Fungal MAGs recovered by fMEM also resolved potentially clinically-relevant genes, not fully predictable from taxonomy alone and revealed genomic content absent from currently-available same-species reference genomes. fMEM is compatible with a whole-genome amplification (including long-read sequencing workflows). Long reads from fMEM-processed samples provided high coverage (>10X) over fungal assemblies. fMEMs compatibility with long-read sequencing enables recovery of genes that would be difficult to assemble with short reads alone. fMEM may enable new insights into the role of human-associated fungi, impacting public health, clinical management, and research into complex diseases with suspected fungal roles. ImportanceFungi influence human health, infectious disease, and the microbiome, but direct genome analysis from clinical samples has remained impractical because fungal DNA is often overwhelmed by human DNA. We developed a fungal microbial enrichment method (fMEM) that enables direct-from-sample fungal metagenomic sequencing and genome recovery from bronchoalveolar lavage samples without requiring culture for genome assembly. fMEM recovers genome-level features not predicted by taxonomy or current same-species reference genomes and is compatible with long-read sequencing workflows that can recover loci missed by short-read sequencing. fMEM opens new opportunities for culture-independent fungal genomics, clinical microbiology, comparative genomics, and mechanistic studies of human-associated fungi.

Bacteria often evolve resistance to phage infection by altering the cell-surface structures required for viral adsorption. However, the role extracellular metabolites play in influencing phage susceptibility and the evolution of phage resistance remains unclear. Here, we evaluated whether sustained exposure to putrescine, a polyamine released during phage-mediated cell lysis, alters susceptibility of the pathogen Pseudomonas aeruginosa to the type IV pili-dependent phage DMS3vir. Using adaptive laboratory evolution over [~]66 generations, we evolved P. aeruginosa with or without putrescine and with or without DMS3vir. As expected, direct phage exposure rapidly led to complete phage resistance. Interestingly, populations exposed to putrescine also developed phage resistance by the end of the experiment, despite having never encountered the phage. Whole-population genome sequencing revealed parallel mutations in genes associated with type IV pili and the global transcriptional regulator mexT. Using transposon insertion mutants in the type IV ATPases pilT and pilB, we confirmed that disruption of these genes leads to DMS3vir phage resistance. We also used a type IV pilus biogenesis factor fimV transposon mutant, which showed a putrescine-dependent reduction in phage susceptibility. These findings show that sustained exposure to a host-derived metabolite can drive the evolution of phage resistance through modification of key phage-adsorption sites and regulatory genes. Our work identifies elevated polyamine exposure as a selective pressure that promotes type IV pili-mediated phage resistance, even in the absence of phage exposure. IMPORTANCEPseudomonas aeruginosa is a major cause of hospital-acquired infections and a key priority for phage-based therapies. Previous work has shown that the polyamine putrescine is released into the extracellular environment during cell lysis. These signals can then transiently reduce susceptibility to bacteriophage infection and alert neighboring cells to danger. Our research demonstrates that long-term exposure to putrescine can drive heritable phage resistance without prior exposure to phage. We show that resistance is linked to mutations in genes involved in type IV pili assembly. This work further demonstrates the critical role that polyamines can play in promoting phage resistance in bacterial communities.

To establish infection and cause disease, the intracellular pathogen Shigella must successfully navigate a series of host defenses and distinct microenvironments within the human body. One way Shigella navigates these enviroments is by using the secondary messenger c-di-GMP, which regulates many different bacterial behaviours. C-di-GMP is synthesized by diguanylate cyclases (DGCs) and broken down by c-di-GMP specific phosphodiesterases (PDEs). In this study, we investigated how Shigellas c-di-GMP specific PDEs impact c-di-GMP turn-over and subsequently biofilm and virulence phenotypes. We knocked out each of Shigellas six c-di-GMP specific PDEs to determine how these PDEs impact biofilm, virulence and c-di-GMP levels within the bacterial cell. We found that these PDEs negatively regulate c-di-GMP levels while modulating Shigellas virulence and biofilm behaviour. We also noted that altering expression of these Shigella PDEs changes bacterial cell size. Transcriptome analysis revealed that a Shigella {Delta}pdeB strain showed reduced expression of many genes, including the virulence genes ipgD and ipgE, as well as genes associated with lipid metabolism. We confirmed that a Shigella {Delta}pdeB strain had altered levels of stearic acid, and expression of pdeB alters Shigella antibiotic susceptibility. This study highlights the complexities of c-di-GMP signaling in regulating numerous Shigella pathways.

Cystic fibrosis (CF) is a multisystem disease characterized by the accumulation of mucus in the airways that promotes pathogen colonization, leading to respiratory exacerbations, lung failure, and death. Culture-independent approaches have revealed that the CF airway harbors a complex microbiota, including opportunistic pathogens and bacteria that colonize the oropharynx. Here, we reanalyzed 5,260 16S rRNA gene microbiota datasets to infer ecological associations between members of the CF microbiota. We determined that pathogens are more likely to proliferate and dominate when present, while oropharyngeal bacteria are more likely to form persistent communities. Further, we found higher diversity and increasing numbers of inferred interactions were positively associated with lung function. In contrast, pathogens were negatively associated both with each other and with oropharyngeal bacteria, suggesting that they may disrupt the microbiota. To validate these predictions, we cultured 1,597 bacterial isolates from 96 people with CF and performed 12,542 coculture assays against eight representative CF pathogenic and oropharyngeal bacteria. 23% of these interactions resulted in growth inhibition. While Pseudomonas isolates were, on average, the most inhibitory, we observed variable activity among isolates. We then confirmed that Pseudomonas aeruginosa isolates, even those from the same donor and timepoint, exhibited significant differences in their metabolome and bioactivity profiles that correlated with acquisition of mutations. Together, our results suggest that pathogens may disrupt the CF microbiota and bloom in part through differential metabolite production. Furthermore, these data highlight that characterizing multiple isolates is necessary to capture the full landscape of chemically mediated interactions within microbial communities. ImportanceThe cystic fibrosis (CF) airway harbors a complex microbiota, including oropharyngeal bacteria and opportunistic pathogens that establish chronic infections and cause lung failure. We confirmed that microbiota diversity is correlated with health and that a pathogen-dominated microbiota is associated with reduced lung function. We then inferred microbial interactions, which suggested that pathogens are able to disrupt the microbiota. To validate these predictions, we cultured bacterial isolates from people with CF and performed thousands of coculture assays, finding that approximately one-quarter of interactions resulted in growth inhibition. Pseudomonas broadly inhibited other members of the CF airway microbiota. However, we observed marked variability in bioactivity and metabolite profiles of Pseudomonas aeruginosa isolates, even from the same donor at the same time. Our results suggest that pathogens disrupt the CF microbiota, possibly through bioactive metabolite production, and that characterizing multiple isolates is necessary to capture the complete picture of interactions in these communities.

Expression of a fungal-specific sub-telomeric gene, hrmA, in Aspergillus fumigatus is important for a colony biofilm morphology termed H-MORPH, increased hypoxic fitness, and virulence in a murine model of invasive pulmonary aspergillosis (IPA). How expression of hrmA contributes to virulence and worse disease progression is ill-defined. Increased hrmA expression results in reduced attachment of the extracellular matrix (ECM) to the fungal cell wall resulting in decreased strain adherence. Fungal strains that are less adherent in vitro are typically less virulent as the ECM heteropolysaccharide galactosaminogalactan (GAG) aids in adhesion to host cells and confers protection from host responses. Here we report that the UDP-glucose 4-epimerase encoding gene required for GAG biosynthesis, uge3, is necessary for full virulence of the H-MORPH strain, hrmAREV (AF293::hrmAD304G). In contrast, loss of uge3 in the reference strain AF293 did not significantly impact virulence in the tested IPA murine model. Phenotypic, transcriptomic, and metabolic analyses of uge3 loss in the respective strain backgrounds revealed a key role for Uge3 in central carbon metabolism in a strain specific context that promotes disease progression. These results complement the known role of Uge3 in GAG biosynthesis and highlight strain specific metabolic differences in pathogenic A. fumigatus strains. IMPORTANCEAspergillus fumigatus forms adherent biofilms that contribute to its ability to persist and cause disease. However, significant strain diversity exists with regard to the morphology of A. fumigatus biofilms. A distinct colony morphotype associated with increased disease progression and low oxygen fitness, termed H-MORPH, was recently described. An additional defining feature of the H-MORPH biofilm morphotype is reduced in vitro adherence to surfaces. While reduced fungal strain adherence is most commonly associated with reductions in virulence, H-MORPH strains exhibit increased virulence relative to the well-studied N-MORPH reference strain AF293. Here we discover that the UDP-glucose 4-epimerase, Uge3, plays an important role in H-MORPH central carbon metabolism complementary to its role in production of the extracellular matrix polysaccharide galactosaminogalactan (GAG). In H-MORPH strains, this metabolic role for Uge3 becomes central to virulence. These data highlight A. fumigatus strain specific mechanisms of fungal carbon metabolism related to biofilm matrix production and fungal virulence.

Probiotics are widely used in food animal production to support gut health and immune function, but the indicators of probiotic efficacy and the conditions under which they translate to host benefit remain unclear. Microbiota composition is the most accessible data supporting probiotic effects, yet whether compositional change reliably predicts host outcomes is not well understood. We investigated this question in broiler chickens fed two nutritionally similar basal diets, with or without a commercial probiotic. Microbiota composition was profiled across 6 gastrointestinal regions using 16S rRNA sequencing. To assess systemic functional effects, an in vitro assay building on prior observations of elevated circulating immune cell ATP in probiotic-fed animals was developed. In this assay, serum from each treatment group was applied to a chicken T-lymphocyte cell line before ATP quantitation. Basal diet was the primary driver of microbial community structure, with probiotic-induced compositional shifts observed predominantly in one diet context but minimally in the other. Despite this difference, serum from probiotic-supplemented animals increased T-lymphocyte ATP production across both diets, supporting prior findings and revealing a systemic immunometabolic response independent of broad microbiota restructuring. Functional predictions revealed enrichment of pathways related to mevalonate and carbohydrate metabolism in probiotic-supplemented birds within the more responsive diet context, driven largely by Lactobacillaceae family taxa. These findings demonstrate that basal diet modulates the detectability and nature of probiotic effects on the microbiota, but not the physiological host response. This disconnect has implications for how probiotic efficacy is evaluated and for microbiome targeted interventions across species. ImportanceProbiotics are used widely in food animal production to support gut health and immune function, yet predicting which probiotic preparations will produce meaningful effects remains a challenge. Microbiota composition, profiled by 16S rRNA sequencing, is the most accessible measure of probiotic activity, but it captures only one aspect of the host-microbe dynamic. These data demonstrate that probiotic-induced compositional changes vary substantially between basal diets, while the host immunometabolic response is consistent across diets, demonstrating that compositional readouts alone cannot reliably predict host outcomes. The findings have practical implications for how probiotic efficacy is evaluated and inform the broader effort to design microbiome targeted interventions across both veterinary and human contexts.

Motility of Plasmodium sporozoites (SPZs) in the skin is a key determinant of successful host infection. Earlier studies have described rapid movement of both murine and human SPZs in skin following syringe inoculation. It is typical to classify SPZ trajectories into ``motile'' and ``immotile'' and restrict the analysis of movement patterns to motile SPZs. Because criteria to define motile SPZs are dependent on the study and are often qualitative, it remains unclear if sub-selection of motile tracks introduces biases in characterization of SPZ movement in vivo. We processed imaging data (22 movies) from a recent study of movement of P. falciparum (Pf) and P. yoelii (Py) SPZ in skin. We proposed a novel metric -- maximal spatial spread (MSS or S) --- that is the maximum Euclidean distance between any two recorded positions in a trajectory. We used MSS to classify SPZ trajectories as immotile (S<Sthreshold) or motile (S>Sthreshold) for a given threshold value Sthreshold. Larger Sthreshold values naturally resulted in a smaller fraction of tracks classified as motile, and subsequently, in an increased overall displacement, instantaneous and mean speeds, decreased mean turning angle, and higher initial slopes of the mean squared displacement (MSD) curves. We found that at intermediate values of Sthreshold Pf SPZs had a lower average speed than Py SPZs suggesting that host environment may impact SPZ movement. Both species exhibited a small but statistically significant decline in average speed with time after inoculation but this was also dependent on the Sthreshold value. Our analysis of MSD curves and turning angle distributions suggests that both Pf and Py SPZs undergo correlated random walks -- a type of Brownian walk with short-term superdiffusive displacement. By using a novel methodology of hidden Markov models (moveHMM package in R) we found that SPZ movement is best described by three movement states; however, none of these states corresponded to previously described circling gliding. Taking together, our results suggest that inference of SPZ movement patterns depends on the criteria used to define tracks as motile or immotile. Standardized preprocessing criteria are therefore important when comparing motility across Plasmodium species, experimental time points, or laboratories. Analysis of turning angle distributions and application of hidden Markov models provided additional metrics to quantify distinct modes of SPZ movement in vivo.

Nakaseomyces glabratus (Candida glabrata) is a WHO high-priority fungal pathogen associated with fungal antimicrobial resistance (fAMR). Given nosocomial transmission occurs sporadically, resistant strains could be transmitted, a concern for critically ill patients. We conducted a genomic investigation and retrospective observational study of N. glabratus to identify any nosocomial transmission of fAMR and understand resistance mechanisms and clinical and demiological factors among patients at a quaternary hospital in Melbourne, Australia. We selected stored N. glabratus with and without fAMR associated with similar patient clinical characteristics and performed whole genome sequencing. Clinical and epidemiological data were extracted from medical records. Phylogenetic, mutational, copy-number variation (CNV) and mitochondrial genomic analyses were performed, with a focus on the fAMR gene PDR1. Of 54 isolates collected over seven years, 20 (37%) were fluconazole-resistant and four (7%) had elevated flucytosine minimum inhibitory concentrations (MICs) (range 2-32 g/ml). There were no significant clinical differences between patients with and without fluconazole resistance. Most (55%) fluconazole-resistant isolates carried PDR1 mutations. Resistance was distributed throughout the phylogeny suggesting predominantly independent acquisition. However, a cluster of four resistant isolates with the same PDR1 mutation suggested nosocomial transmission. One probable ERG11 gene duplication, and two petite variants with apparent mitochondrial genomic deletions, were seen in association with fluconazole resistance. In this study, we identified a small probable nosocomial fAMR transmission cluster, and novel variants in PDR1, ERG11 and FCY2 associated with fAMR phenotypes. Future study should confirm functional impacts and systematically investigate for nosocomial transmission of resistance, including colonisation states.

The fungal pathogen Cryptococcus neoformans contains approximately 200 {micro}g of {beta}-1,3-glucan (1,3BG) per 1 mg of dry cell weight when grown under standard culture conditions (YPD medium at 30{degrees}C under aerobic conditions). However, 1,3BG exposure is tightly suppressed, even in capsule-deficient strains, allowing the fungus to evade recognition by the immune receptor dectin-1 and anti-1,3BG antibodies. Although other pathogenic fungi mask 1,3BG with -1,3-glucan (1,3AG) to evade dectin-1 recognition, the factors responsible for 1,3BG masking and dectin-1 evasion in C. neoformans remain incompletely understood. To identify capsule-independent 1,3BG masking and dectin-1 evasion factors, we generated a series of cell wall-related gene deletion strains in the capsule-deficient strain cap59{Delta} using CRISPR/Cas9 and screened for mutants that failed to evade dectin-1 binding. We found eight deletants (cap59{Delta}/mpk1{Delta}, cap59{Delta}/chs3{Delta}, cap59{Delta}/kre5{Delta}, cap59{Delta}/crz1{Delta}, cap59{Delta}/kre6{Delta}/skn1{Delta}, cap59{Delta}/hxl1{Delta}, cap59{Delta}/uge1{Delta}, and cap59{Delta}/ugt1{Delta}) that exhibited increased binding to dectin-1 and/or anti-1,3BG antibody. Since a similar phenotype was not observed in cap59{Delta}/ags1{Delta}, 1,3AG-mediated masking of 1,3BG appears to play a limited role in C. neoformans. These eight deletants induced significantly greater secretion of IL-6 and IL-1{beta} from dendritic cells (DCs) than cap59{Delta} or cap59{Delta}/ags1{Delta}. This enhanced inflammatory response was markedly attenuated in dectin-1-deficient DCs, indicating that the increased immunogenicity was driven by 1,3BG exposure and subsequent dectin-1 recognition. Collectively, these findings demonstrate that multiple genes involved in maintaining cell wall integrity, including those involved in {beta}-1,6-glucan and chitosan biosynthesis, are essential for regulating 1,3BG exposure and enabling C. neoformans to evade dectin-1-mediated immune recognition. HighlightsO_LINovel capsule-independent {beta}-1,3-glucan masking genes in Cryptococcus neoformans were identified. C_LIO_LIThe deletants of these genes displayed higher dectin-1 deposition, contrary to the parental capsule-deficient mutant cap59{Delta}. C_LIO_LIDeletion of these genes led to enhanced secretion of IL-6 and IL-1{beta} from dendritic cells. C_LIO_LIThe enhanced cytokine response was suppressed in dendritic cells lacking dectin-1. C_LIO_LIThese deletant strains have potential to serve as new whole-cell antigens for cryptococcal vaccine development. C_LI

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.

BackgroundMultiple Sclerosis (MS) is a chronic autoimmune disorder characterized by inflammation and demyelination in central nervous system (CNS). Although increasing evidence suggests that gut microbial dysbiosis contributes to MS pathogenesis through the microbiota-gut-brain axis, reproducible microbial signatures associated with disease progression across independent clinical cohorts remain incompletely characterized. ObjectiveThis study aimed to identify conserved gut microbial alterations associated with Multiple Sclerosis by integrating publicly available human gut microbiome datasets and characterizing disease-associated microbial signatures linked to immune dysregulation. DesignHuman gut metagenomic 16S rRNA sequencing data from MS patients and healthy controls obtained from publicly available repositories (NCBI, Bioproject). Raw sequencing reads were processed using a standardized microbiome analysis workflow, including quality control, denoising, taxonomic assignment, phylogenetic reconstruction, diversity analyses, and differential abundance testing. Microbial community structure was evaluated using alpha- and beta-diversity analyses, while statistically significant differences between study groups were assessed using PERMANOVA, Kruskal-Wallis, and ANCOM to identify disease-associated bacterial taxa. ResultsIntegration of independent cohorts revealed consistent alterations in the gut microbial composition of MS patients compared with healthy controls. Significant reductions in microbial diversity and distinct microbial community structures were observed in MS. Differential abundance analysis demonstrated enrichment of the pro-inflammatory family Streptococcaceae, whereas beneficial short-chain fatty acid-producing taxa, particularly Lachnospiraceae, were significantly depleted in MS patients. These conserved microbial alterations indicate disruption of immune-regulatory bacterial communities and support the involvement of gut microbial dysbiosis in MS-associated neuroinflammation. ConclusionThis study identifies a reproducible gut microbial dysbiosis signature associated with Multiple Sclerosis, characterized by expansion of pro-inflammatory bacterial taxa and depletion of beneficial SCFA-producing microorganisms. These findings strengthen the evidence supporting the microbiota-gut-brain axis in MS pathogenesis and highlight microbial community signatures that may contribute to future biomarker development and microbiome- based therapeutic strategies.

Bacteria routinely exhibit unexpected phenotypic and molecular changes in response to spaceflight and spaceflight-analogue conditions, yet the mechanisms by which they sense and respond to these low fluid shear environments are not fully elucidated. We previously demonstrated that spaceflight and low shear modeled microgravity (LSMMG) altered motility and chemotaxis gene expression in Salmonella enterica serovar Typhimurium (S. Typhimurium), raising the possibility that flagella mediate responses of the pathogen to these environments. Herein, we investigated whether LSMMG culture alters S. Typhimurium motility and examined the role of flagella in regulating pathogenesis-associated stress and infection phenotypes. LSMMG enhanced the swimming motility of wild-type S. Typhimurium relative to 1xg controls; a trend which persisted even in the absence of the global stress response regulators Hfq and RpoS. This finding was unexpected, as {Delta}hfq mutants are typically defective for motility under conventional culture conditions. Motility was also observed in the flagella-deficient {Delta}flhDC mutant following LSMMG and 1xg culture, although the relative motility pattern differed relative to wild-type. Collectively, these results indicate that flagella contribute to LSMMG-enhanced motility, but are not strictly required under these conditions. Conditioned supernatant exchange demonstrated that LSMMG-induced motility changes are cell-intrinsic rather than mediated by extracellular factors. While flagella were dispensable for many pathogenesis-related phenotypes tested, their deletion selectively altered the magnitude of LSMMG-associated thermal stress and intracellular survival in human intestinal epithelial cells. Together, these findings demonstrate that motility and pathogenesis-related responses in S. Typhimurium are governed by multiple regulatory pathways that differentially respond to LSMMG and 1xg conditions. IMPORTANCESpaceflight and spaceflight-analogue conditions alter bacterial physiology in unexpected ways that are important for pathogenesis, yet the mechanisms by which bacteria sense and respond to low fluid shear environments remain incompletely understood. This study shows that low shear modeled microgravity (LSMMG) enhances Salmonella Typhimurium motility and produces unexpected motility phenotypes in mutants lacking Hfq or the flagellar master regulator FlhDC. These findings indicate that flagellar biosynthesis contributes to LSMMG-enhanced motility but is not strictly required for motility under these conditions. We also suggest that flagella influence the magnitude of selected stress and infection phenotypes rather than serving as an absolute requirement for LSMMG responsiveness. Together, these results highlight the complexity of bacterial mechanotransduction under simulated microgravity conditions and advances our understanding of how a foodborne pathogen adapts to physiological low fluid shear environments encountered both in space and during terrestrial infection of the intestinal tract.