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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. 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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3. 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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4. Phenotypic tolerance for rDNA copy number variation within the natural range of C. elegans

   https://doi.org/10.1371/journal.pgen.1011759  

   Hall, Ashley N; Morton, Elizabeth A; Walters, Rebecca; Cuperus, Josh T; Queitsch, Christine (July 2025, PLOS Genetics)

   SARINAY_CENIK, ELIF (Ed.)

   The genes for ribosomal RNA (rRNA) are encoded by ribosomal DNA (rDNA), whose structure is notable for being present in arrays of tens to thousands of tandemly repeated copies in eukaryotic genomes. The exact number of rDNA copies per genome is highly variable within a species, with differences between individuals measuring in potentially hundreds of copies and megabases of DNA. The extent to which natural variation in rDNA copy number impacts whole-organism phenotypes such as fitness and lifespan is poorly understood, in part due to difficulties in manipulating such large and repetitive tracts of DNA even in model organisms. Here, we used the natural resource of copy number variation in C. elegans wild isolates to generate new tools and investigated the phenotypic consequences of this variation. Specifically, we generated a panel of recombinant inbred lines (RILs) using a laboratory strain derivative with ~130 haploid rDNA copies and a wild isolate with ~417 haploid rDNA copies, one of the highest validated C. elegans rDNA copy number arrays. We find that rDNA copy number is stable in the RILs, rejecting prior hypotheses that predicted copy number instability and copy number reversion. To isolate effects of rDNA copy number on phenotype, we produced a series of near isogenic lines (NILs) with rDNA copy numbers representing the high and low end of the rDNA copy number spectrum in C. elegans wild isolates. We find no correlation between rDNA copy number and phenotypes of rRNA abundance, competitive fitness, early life fertility, lifespan, or global transcriptome under standard laboratory conditions. These findings demonstrate a remarkable ability of C. elegans to tolerate substantial variation in a locus critical to fundamental cell function. Our study provides strain resources for future investigations into the boundaries of this tolerance. 

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5. Evolutionary history and association with seaweeds shape the genomes and metabolisms of marine bacteria

   https://doi.org/10.1128/msphere.00996-24  

   Pfister, Catherine A; Berlinghof, Johanna; Bogan, Maximiliana; Cardini, Ulisse; Gobet, Angélique; Hamon-Giraud, Pauline; Hart, Jessica; Jimenez, Natalia; Siegel, Anne; Stanfield, Emma; et al (June 2025, mSphere)

   Campbell, Barbara J (Ed.)

   ABSTRACT Seaweeds harbor a rich diversity of bacteria, providing them with metabolic resources and a surface for attachment and biofilm development. The host’s unique environment potentially shapes the bacterial genomes and promotes adaptations for a symbiotic lifestyle. To investigate whether the genomes of seaweed-associated bacteria are genetically and metabolically distinct from their close free-living relatives in seawater, we compared both the seaweed-associated and free-living counterparts of 72 bacterial genera across 16 seaweed hosts using whole-genome sequences or high-quality metagenome-assembled genomes. While taxonomic affiliation strongly influenced genome characteristics such as GC content, gene number, and size, host association had a lower effect overall. A reduced genome size was suggested only inNereocystis luetkeana-associated microbes, while onlyAscophyllum nodosum-associated bacteria had an increased GC content. Metabolic adaptations were indicated from the genomes of seaweed-associated bacteria, including enriched pathways for B vitamin production, complex carbohydrate utilization, and amino acid biosynthesis. In particular,Flavobacteriiashowed the most pronounced differences between host-associated and free-living strains. We further hypothesized that bacteria associated with seaweed might have evolved to complement their host’s metabolism and tested this inference by analyzing the genomes of both the seaweedEctocarpus subulatusand its 28 bacterial associates but found no evidence for such complementarity. Our analyses of 72 paired bacterial genomes highlighted significant metabolic differences in seaweed-associated strains with implications for carbon, nitrogen, and sulfur cycling in the coastal ocean. IMPORTANCEWe hypothesized that the unique environment of seaweeds in coastal oceans shapes bacterial genomes and promotes a symbiotic lifestyle. We compared the genomes of bacteria isolated from seaweed with bacteria from the same genus found free-living in seawater. For genome features that included the number of genes, the size of the genome, and the GC content, taxonomy was of greater importance than bacterial lifestyle. When we compared metabolic abilities, we again found a strong effect of taxonomy in determining metabolism. Although several metabolic pathways differed between free-living and host-associated bacteria, this was especially prominent forFlavobacteriiain the phylumBacteroidota. Notably, bacteria living on seaweeds had an increased occurrence of genes for B vitamin synthesis, complex carbohydrate use, and nitrogen uptake, indicating that bacterial genomes reflect both their evolutionary history and the current environment they inhabit. 

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