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1. Navigating cross-reactivity and host species effects in a serological assay: A case study of the microscopic agglutination test for Leptospira serology

   https://doi.org/10.1371/journal.pntd.0012042  

   Mummah, Riley O; Gomez, Ana_C R; Guglielmino, Angela H; Borremans, Benny; Galloway, Renee L; Prager, Katherine C; Lloyd-Smith, James O (October 2024, PLOS Neglected Tropical Diseases)

   Chang, Yung-Fu (Ed.)

   BackgroundSerology (the detection of antibodies formed by the host against an infecting pathogen) is frequently used to assess current infections and past exposure to specific pathogens. However, the presence of cross-reactivity among host antibodies in serological data makes it challenging to interpret the patterns and draw reliable conclusions about the infecting pathogen or strain. Methodology/Principal findingsIn our study, we use microscopic agglutination test (MAT) serological data from three host species [California sea lion (Zalophus californianus), island fox (Urocyon littoralis), and island spotted skunk (Spilogale gracilis)] with confirmed infections to assess differences in cross-reactivity by host species and diagnostic laboratory. All host species are known to be infected with the same serovar ofLeptospira interrogans. We find that absolute and relative antibody titer magnitudes vary systematically across host species and diagnostic laboratories. Despite being infected by the sameLeptospiraserovar, three host species exhibit different cross-reactivity profiles to a 5-serovar diagnostic panel. We also observe that the cross-reactive antibody titer against a non-infecting serovar can remain detectable after the antibody titer against the infecting serovar declines below detectable levels. Conclusions/SignificanceCross-reactivity in serological data makes interpretation difficult and can lead to common pitfalls. Our results show that the highest antibody titer is not a reliable indicator of infecting serovar and highlight an intriguing role of host species in shaping reactivity patterns. On the other side, seronegativity against a given serovar does not rule out that serovar as the cause of infection. We show that titer magnitudes can be influenced by both host species and diagnostic laboratory, indicating that efforts to interpret absolute titers (e.g., as indicators of recent infection) must be calibrated to the system under study. Thus, we implore scientists and health officials using serological data for surveillance to interpret the data with caution. 

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2. Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity

   https://doi.org/10.1371/journal.ppat.1012047  

   Hodoameda, Peter; Ebel, Gregory D; Mukhopadhyay, Suchetana; Clem, Rollie J (February 2024, PLOS Pathogens)

   Goodman, Alan G (Ed.)

   Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infectedAedes aegyptimosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individualAe.aegyptimosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles. 

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3. Host-mimicking conditions promote Pseudomonas aeruginosa PA14 virulence gene expression

   https://doi.org/10.3389/fmicb.2025.1557664  

   Grace, Amber; Sahu, Rajnish; Owen, Donald R; Dennis, Vida A (April 2025, Frontiers in Microbiology)

   BackgroundPseudomonas aeruginosais a ubiquitous, opportunistic bacterium whose highly plastic genome and adaptable phenotype have yielded serious treatment challenges for immunocompromised patients. Antibiotic alternatives, such as anti-virulence therapeutics, have gained interest because they disable bacterial virulence mechanisms, thereby restoring the killing efficacy of host immunity or traditional antibiotics. Identifying successful anti-virulence therapeutics may require a paradigm shift from the decades-old antimicrobial susceptibility testing (AST) in Mueller Hinton broth to media that foster optimal virulence expression. MethodsThis study evaluates the virulence gene expression and activity ofP. aeruginosaPA14 in host-mimicking conditions, represented by Dulbecco’s Modified Eagle’s Medium (DMEM) without serum, with fetal bovine serum (FBS), or with human serum (HuS) in comparison to standard antimicrobial susceptibility testing conditions, represented by Cation-adjusted Mueller Hinton broth (CAMHB). PA14 twitching motility and pyoverdine production were evaluated under these conditions. ResultsFor the first time, our study reveals that culturing the highly virulentP. aeruginosaPA14 in host-mimicking media enhances the expression of multiple virulence therapeutic targets that are critical to host colonization and infection. RNA sequencing showed that multiple Type III Secretion (T3SS), Type I Secretion (T1SS), pyoverdine biosynthesis, uptake and efflux, and Type IV pili (T4P) initiation genes were promoted when PA14 was transitioned into host-mimicking conditions but remained unchanged when transitioned into standard AST conditions. Moreover, qPCR results disclosed that HuS and FBS delivered differential effects on the expression of membrane-associated virulence genes involved in host colonization. Our macroscopic PA14 twitching motility results aligned more closely with PA14 growth patterns than with virulence gene expression patterns. Our microtiter biofilm assay, however, revealed earlier biofilm formation in DMEM 0 than in AST conditions and both showed inhibited twitching motility in serum conditions. UV-Vis spectra showed that pyoverdine production aligned with our gene expression data, revealing higher pyoverdine production in serum conditions for planktonic PA14. DiscussionOverall, our findings support using host-mimicking conditions to improve the expression of candidate targets for anti-virulence therapeutics againstP. aeruginosaPA14 in a planktonic state. These recommendations may be broadly applicable for antivirulence therapeutic screening against multiple bacterial species at large. 

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4. Dense time-course gene expression profiling of the Drosophila melanogaster innate immune response

   https://doi.org/10.1186/s12864-021-07593-3  

   Schlamp, Florencia; Delbare, Sofie_Y_N; Early, Angela_M; Wells, Martin_T; Basu, Sumanta; Clark, Andrew_G (April 2021, BMC Genomics)

   Abstract BackgroundImmune responses need to be initiated rapidly, and maintained as needed, to prevent establishment and growth of infections. At the same time, resources need to be balanced with other physiological processes. On the level of transcription, studies have shown that this balancing act is reflected in tight control of the initiation kinetics and shutdown dynamics of specific immune genes. ResultsTo investigate genome-wide expression dynamics and trade-offs after infection at a high temporal resolution, we performed an RNA-seq time course onD. melanogasterwith 20 time points post Imd stimulation. A combination of methods, including spline fitting, cluster analysis, and Granger causality inference, allowed detailed dissection of expression profiles, lead-lag interactions, and functional annotation of genes through guilt-by-association. We identified Imd-responsive genes and co-expressed, less well characterized genes, with an immediate-early response and sustained up-regulation up to 5 days after stimulation. In contrast, stress response and Toll-responsive genes, among which were Bomanins, demonstrated early and transient responses. We further observed a strong trade-off with metabolic genes, which strikingly recovered to pre-infection levels before the immune response was fully resolved. ConclusionsThis high-dimensional dataset enabled the comprehensive study of immune response dynamics through the parallel application of multiple temporal data analysis methods. The well annotated data set should also serve as a useful resource for further investigation of theD. melanogasterinnate immune response, and for the development of methods for analysis of a post-stress transcriptional response time-series at whole-genome scale. 

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5. Mitochondrial genome diversity across the subphylum Saccharomycotina

   https://doi.org/10.3389/fmicb.2023.1268944  

   Wolters, John F.; LaBella, Abigail L.; Opulente, Dana A.; Rokas, Antonis; Hittinger, Chris Todd (November 2023, Frontiers in Microbiology)

   IntroductionEukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeastSaccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available. MethodsBy extracting mitochondrial assemblies from existing short-read genome sequence datasets, we have greatly expanded both the number of available genomes and the coverage across sparsely sampled clades. ResultsComparison of 353 yeast mitochondrial genomes revealed that, while size and GC content were fairly consistent across species, those in the generaMetschnikowiaandSaccharomycestrended larger, while several species in the order Saccharomycetales, which includesS. cerevisiae, exhibited lower GC content. Extreme examples for both size and GC content were scattered throughout the subphylum. All mitochondrial genomes shared a core set of protein-coding genes for Complexes III, IV, and V, but they varied in the presence or absence of mitochondrially-encoded canonical Complex I genes. We traced the loss of Complex I genes to a major event in the ancestor of the orders Saccharomycetales and Saccharomycodales, but we also observed several independent losses in the orders Phaffomycetales, Pichiales, and Dipodascales. In contrast to prior hypotheses based on smaller-scale datasets, comparison of evolutionary rates in protein-coding genes showed no bias towards elevated rates among aerobically fermenting (Crabtree/Warburg-positive) yeasts. Mitochondrial introns were widely distributed, but they were highly enriched in some groups. The majority of mitochondrial introns were poorly conserved within groups, but several were shared within groups, between groups, and even across taxonomic orders, which is consistent with horizontal gene transfer, likely involving homing endonucleases acting as selfish elements. DiscussionAs the number of available fungal nuclear genomes continues to expand, the methods described here to retrieve mitochondrial genome sequences from these datasets will prove invaluable to ensuring that studies of fungal mitochondrial genomes keep pace with their nuclear counterparts. 

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