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The bacterial genus Acinetobacter includes species found in environmental habitats like soil and water, as well as taxa adapted to be host-associated or pathogenic. High genetic diversity may allow for this habitat flexibility, but the specific genes underlying switches between habitats are poorly understood. One lineage of Acinetobacter has undergone a substantial habitat change by evolving from a presumed soil-dwelling ancestral state to thrive in floral nectar. Here, we compared the genomes of floral-dwelling and pollinator-associated Acinetobacter, including newly described species, with genomes from relatives found in other environments to determine the genomic changes associated with this ecological shift. Following one evolutionary origin of floral nectar adaptation, nectar-dwelling Acinetobacter taxa have undergone reduction in genome size compared with relatives and have experienced dynamic gene gains and losses as they diversified. Gene content changes suggest a shift to metabolism of monosaccharides rather than diverse carbohydrates, and scavenging of nitrogen sources, which we predict to be beneficial in nectar environments. Gene gains appear to result from duplication events, evolutionary divergence, and horizontal gene transfer. Most notably, nectar-dwelling Acinetobacter acquired the ability to degrade pectin from plant pathogens, and the genes underlying this ability have duplicated and are under selection within the clade. We hypothesize that this ability was a key trait for adaptation to floral nectar, as it could improve access to nutrients in the nutritionally unbalanced habitat of nectar. These results identify the genomic changes and traits coinciding with a dramatic habitat switch from soil to floral nectar.

Importance: Many bacteria, including the genus Acinetobacter, commonly evolve to exploit new habitats. However, the genetic changes that underlie habitat switches are often unknown. Floral nectar is home to specialized microbes that can grow in this nutritionally unbalanced habitat. Several specialized Acinetobacter species evolved from soil-dwelling relatives to become common and abundant in floral nectar. Here, we investigate the genomic adaptations required to successfully colonize a novel habitat like floral nectar. We performed comparative genomics analyses between nectar-dwelling Acinetobacter and Acinetobacter species from other environments, like soil and water. We find that although gene loss coincided with the switch to living in nectar, gains of specific genes from other bacteria may have been particularly important for this ecological change. Acinetobacter living in nectar gained genes for degrading pectin, a plant polysaccharide, which may improve access to nutrients in their environment. These findings shed light on how evolutionary novelty evolves in bacteria.

The human microbiome, the community of microorganisms that reside on and inside the human body, is critically important for health and disease. However, it is influenced by various factors and may vary among individuals residing in distinct geographic regions. In this study, 220 samples, consisting of sterile swabs from palmar skin and oral and nasal cavities were collected from Chinese Han individuals living in Shanghai, Chifeng, Kunming, and Urumqi, representing the geographic regions of east, northeast, southwest, and northwest China. The full-length 16S rRNA gene of the microbiota in each sample was sequenced using the PacBio single-molecule real-time sequencing platform, followed by clustering the sequences into operational taxonomic units (OTUs). The analysis revealed significant differences in microbial communities among the four regions. Cutibacterium was the most abundant bacterium in palmar samples from Shanghai and Kunming, Psychrobacter in Chifeng samples, and Psychrobacillus in Urumqi samples. Additionally, Streptococcus and Staphylococcus were the dominant bacteria in the oral and nasal cavities. Individuals from the four regions could be distinguished and predicted based on a model constructed using the random forest algorithm, with the predictive effect of palmar microbiota being better than that of oral and nasal cavities. The prediction accuracy using hypervariable regions (V3-V4 and V4-V5) was comparable with that of using the entire 16S rRNA. Overall, our study highlights the distinctiveness of the human microbiome in individuals living in these four regions. Furthermore, the microbiome can serve as a biomarker for geographic origin inference, which has immense application value in forensic science.IMPORTANCEMicrobial communities in human hosts play a significant role in health and disease, varying in species, quantity, and composition due to factors such as gender, ethnicity, health status, lifestyle, and living environment. The characteristics of microbial composition at various body sites of individuals from different regions remain largely unexplored. This study utilized single-molecule real-time sequencing technology to detect the entire 16S rRNA gene of bacteria residing in the palmar skin, oral, and nasal cavities of Han individuals from four regions in China. The composition and structure of the bacteria at these three body sites were well characterized and found to differ regionally. The results elucidate the differences in bacterial communities colonizing these body sites across different regions and reveal the influence of geographical factors on human bacteria. These findings not only contribute to a deeper understanding of the diversity and geographical distribution of human bacteria but also enrich the microbiome data of the Asian population for further studies.

Prominent virulence traits of Candida albicans include its ability to produce filamentous hyphal cells and grow as a biofilm. These traits are under control of numerous transcription factors (TFs), including Brg1 and Rme1. In the reference strain SC5314, a brg1Δ/Δ mutant has reduced levels of biofilm/filament production; a brg1Δ/Δ rme1Δ/Δ double mutant has wild-type levels of biofilm/filament production. Here, we asked whether this suppression relationship is preserved in four additional strain backgrounds: P76067, P57055, P87, and P75010. These strains represent diverse clades and biofilm/filament production abilities. We find that a rme1Δ/Δ mutation restores biofilm/filament production in a brg1Δ/Δ mutant of P76067, but not in brg1Δ/Δ mutants of P57055, P87, and P75010. We speculate that variation in activities of two functionally related TFs, Nrg1, and Ume6, may cause the strain-limited impact of the rme1Δ/Δ mutation.

Importance: Candida albicans is a widespread fungal pathogen. The regulatory circuitry underlying virulence traits is well studied in the reference strain background, but not in other clinical isolate backgrounds. Here, we describe a pronounced example of strain variation in the control of two prominent virulence traits, biofilm formation and filamentation.

Measuring the immunogenicity of pneumococcal vaccines involves the use of immunoassays to measure serotype-specific immunoglobulin G (IgG) antibody levels post-vaccination with the current Streptococcus pneumoniae human reference serum standard (007sp) for anti-pneumococcal capsule antibodies. Development of new pneumococcal conjugate vaccines (PCVs) with additional serotypes not in 007sp (e.g., V116, a 21-valent PCV) requires a new reference serum. Antibody concentrations to 33 pneumococcal serotypes were assigned in a new Merck Pneumococcal Reference Serum Standard (MPRSS-01; Merck Pneumococcal Reference Serum Standard) using the pneumococcal electrochemiluminescence assay. MPRSS-01 was generated by pooling high-titer serum samples from adults immunized with either 23-valent pneumococcal polysaccharide vaccine (PPSV23) or V116. For the 24 serotypes with established IgG concentrations, the corresponding antibody concentrations in MPRSS-01 were assigned via direct calibration to 007sp, while a cross-standardization approach was used for the nine novel serotypes. Serotype 7F was initially chosen as the reference calibrator for cross-standardization due to parallelism across dilution-response curves demonstrated across all 33 serotypes, and an evaluation of the single-calibrator approach was conducted for the 24 serotypes. Potential systematic bias from using a unique serotype for calibration was identified and addressed by further adjusting the estimated IgG concentrations of the nine novel serotypes. Using the final MPRSS-01 antibody concentration assignments, and the calibration factor relating MPRSS-01 to 007sp, antibody concentration assignments for 007sp were provided for the nine novel serotypes. This proposal was accepted by the Center for Biologics Evaluation and Research (CBER), enabling V116 to bridge old and new human pneumococcal reference sera.IMPORTANCEImmunogenicity of pneumococcal vaccines is measured using post-vaccination serotype-specific immunoglobulin G (IgG) antibodies in serum using enzyme-linked immunoassays with the 007sp reference serum containing serotype-specific IgG for 24 pneumococcal serotypes. With the development of next-generation PCVs, a new S. pneumoniae reference serum standard was needed to include serotypes beyond the existing 24 in 007sp. In this study, antibody concentrations to 33 pneumococcal serotypes were assigned in a new Merck Pneumococcal Reference Serum Standard (MPRSS-01) using the pneumococcal electrochemiluminescence assay, enabling V116 to maintain the link to the historical human pneumococcal standard reference serum while utilizing the new human pneumococcal reference serum.

The universal bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays critical roles in regulating a variety of bacterial functions such as biofilm formation and virulence. The metabolism of c-di-GMP is inversely controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). Recently, increasing studies suggested that the protein-protein interactions between DGCs/PDEs and their partners appear to be a common way to achieve specific regulation. In this work, we showed that the PDE ProE can interact with PQS quorum sensing protein PqsE to regulate pyocyanin production in Pseudomonas aeruginosa. Our bacterial two-hybrid assay demonstrated that ProE directly interacts with PqsE, and isothermal titration calorimetry and surface plasmon resonance assay further confirmed that the binding affinity of ProE with PqsE is at micromolar level. Both ProE and PqsE negatively regulate intracellular c-di-GMP levels. Furthermore, our transcriptomic study showed that co-expression of ProE and PqsE significantly changes the gene expression profiles in P. aeruginosa, especially with increased expression of pyocyanin genes, and the qPCR and phenotypic results confirmed the transcriptome data. Taken together, our study suggested that the interaction between ProE and PqsE plays a critical role in regulation of pyocyanin production and highlights the importance of protein-protein interaction mediated c-di-GMP signaling in P. aeruginosa.IMPORTANCEc-di-GMP is pivotal in orchestrating various bacterial functions. In Pseudomonas aeruginosa, the nuanced balance of intracellular c-di-GMP is maintained by approximately 41 diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). Emerging studies indicate that the c-di-GMP metabolic DGCs and PDEs may be involved in the signal transduction process by directly binding to the target protein, thus influencing downstream function. Despite their known importance, the precise functions of these proteins, especially their interacting partners, remain unclear. In this study, we identified that PQS quorum sensing system protein PqsE is a binding partner of c-di-GMP phosphodiesterase ProE; further analysis suggested that the ProE specifically interacts with PqsE to promote pyocyanin production. Our study extended the regulatory mechanism of the c-di-GMP signal transduction and quorum sensing in governing bacterial physiology.

The widespread use of azole antifungals in agriculture and clinical settings has led to serious drug resistance. Overexpression of the azole drug target 14α-demethylase ERG11 (CYP51) is the most common fungal resistance mechanism. However, the presence of additional regulatory proteins in the transcriptional response of erg11 is not yet fully elucidated. In this study, leveraging the identified key promoter region of erg11 that controls its response to azoles in Neurospora crassa, we pinpointed a protein, Crf4-3, which harbors a PWWP domain and exerts a positive regulatory influence on azole resistance, as determined by DNA pulldown assays. The removal of Crf4-3 results in heightened sensitivity to azoles while remaining unaffected by other stressors tested. Additionally, the deletion leads to the abolition of transcriptional responses of genes such as erg11 and erg6 to ketoconazole. Interestingly, the basal expression of erg1, erg11, erg25, and erg3A is also affected by the deletion of crf4-3, indicating its role in sterol homeostasis. Crf4-3 homologs are broadly distributed across the Pezizomycotina fungi. The gene deletion for its homologous protein in Aspergillus fumigatus also significantly improves sensitivity to azoles such as voriconazole, primarily through the attenuation of the transcriptional response of erg11. Our data, for the first time, identified Crf4-3 as a novel regulatory protein in the azole stress response of filamentous fungi, offering fresh insights into the mechanisms of azole resistance.IMPORTANCETranscriptional control of pivotal genes, such as erg11, stands as the primary driver of azole resistance. Although considerable effort has been dedicated to identifying transcription factors involved, our knowledge regarding the use of transcriptional regulation strategies to combat azole resistance is currently limited. In this study, we reveal that a PWWP domain-containing protein Crf4-3, which is conserved in Pezizomycotina fungi, modulates fungal azole sensitivity by transcriptionally regulating sterol biosynthetic genes, including erg11. These results also broaden the understanding of fungal PWWP domain-containing proteins regarding their roles in regulating resistance against azole antifungals. Considering research on small molecules targeting the PWWP domain in humans, Crf4-3 homolog emerges as a promising target for designing fungal-specific drugs to combat azole resistance.

Coccidioides spp. are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The Coccidioides host form, the spherule, is unique and highly undercharacterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal medium to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.IMPORTANCECoccidioides spp. are thermally dimorphic fungal pathogens found in the Southwest United States, Mexico, Central America, and South America. Coccidioides can infect both immunocompetent and immunocompromised people and can cause a devastating disseminated infection, including meningitis, with 30% mortality despite all currently available treatments. In this work, we tackle one of the current largest technical barriers to studying the fungus Coccidioides: reliably growing its host form in vitro. Our work is impactful because we have created a set of foundational tools for the burgeoning field of Coccidioides pathogenesis research. We have carefully optimized conditions that allow the development of Coccidioides in vitro into its pathogenic form. This work will open up many lines of investigation into the molecules that underlie Coccidioides pathogenesis.

Klebsiella pneumoniae is a prominent Gram-negative and encapsulated opportunistic pathogen that causes a multitude of infections such as severe respiratory and healthcare-associated infections. Despite the widespread anti-microbial resistance and the high mortality rate, currently, no clinically vaccine is approved for battling K. pneumoniae. To date, messenger RNA (mRNA) vaccine is one of the most advancing technologies and are extensively investigated for viral infection, while infrequently applied for prevention of bacterial infections. In the present study, we aim to construct a new mRNA vaccine encoding YidR or combining with a tissue plasminogen activator signal sequence for preventing K. pneumoniae infection. Adaptive immunity was determined in mRNA vaccines-immunized mice and the protective effects of mRNA vaccines were evaluated in K. pneumoniae infected models. The results showed that lipid nanoparticle (LNP)-YidR-mRNA vaccine was produced with good morphology, high the encapsulation efficiency, and the specific antigen was highly expressed in cells in vitro. In addition, immunization with either LNP-YidR or LNP-YidR-SP elicited a Th1-biased immune response, reduced bacterial load, and provided broad protection in the lung infection models. Importantly, the LNP-YidR-SP mRNA vaccine induced strong adaptive humoral and cellular immunity and increased the survivability of mice compared to the other groups. Our findings serve as a focal point for developing a potential mRNA vaccine against K. pneumoniae, indicating the potential of mRNA vaccines for improving next-generation bacterial vaccine.IMPORTANCEK. pneumoniae is a notorious and clinical bacterium that is evolving in community-acquired and nosocomial settings. This opportunistic pathogen causes severe infectious diseases, including urinary tract infection and pneumonia, and causes a concerning global public burden. Despite efforts having been created to develop different types of K. pneumoniae vaccines, there is no licensed vaccine for preventing K. pneumoniae infection. Therefore, to develop an effective tactic is essential to combat K. pneumoniae-caused diseases. This study provides a novel vaccine strategy against K. pneumoniae and a potent platform to elicit high levels of humoral and cell-meditated immunity.

During aerobic growth, S. aureus relies on acetate overflow metabolism, a process where glucose is incompletely oxidized to acetate, for its bioenergetic needs. Acetate is not immediately captured as a carbon source and is excreted as waste by cells. The underlying factors governing acetate overflow in S. aureus have not been identified. Here, we show that acetate overflow is favored due to a thermodynamic bottleneck in the TCA cycle specifically involving the oxidation of succinate to fumarate by succinate dehydrogenase. This bottleneck reduces flux through the TCA cycle, making it more efficient for S. aureus to generate ATP via acetate overflow metabolism. Additionally, the protein allocation cost of maintaining ATP flux through the restricted TCA cycle is greater than that of acetate overflow metabolism. Finally, we show that the TCA cycle bottleneck provides S. aureus the flexibility to redirect carbon toward maintaining redox balance through lactate overflow when oxygen becomes limiting, albeit at the expense of ATP production through acetate overflow. Overall, our findings suggest that overflow metabolism offers S. aureus distinct bioenergetic advantages over a thermodynamically constrained TCA cycle, potentially supporting its commensal-pathogenic lifestyle.

Honey bees are the third most economically important agricultural animal in the world due to their role as pollinators. Honey bee pollination services and all hive duties are performed by female workers, while the male drones have one job to mate and share their genetics with a virgin queen from another colony. Thus, drone fitness is directly tied to queen success and colony survival, yet they have been severely understudied compared to their female counterparts. In other insects, microbes discovered in the gut and reproductive organs have been shown to be important for reproductive success and/or overall host health. To our knowledge, the existence of microbes in drone reproductive tissues has never been investigated. Moreover, our understanding of the gut microbiota of drones is severely limited, especially when compared to honey bee workers. Here, we sampled conventional drones from healthy colonies and used 16S amplicon sequencing to identify and characterize bacteria in the reproductive organs of immature and mature drones. After identifying bacteria in drone reproductive tissues, we performed a controlled experiment in which newly emerged drones were exposed to different rearing conditions in order to determine when and how they acquire their reproductive and gut microbiota. Overall, we discovered a set of core bacteria in the reproductive and gut tissues of conventionally reared drones and revealed that social interactions are important for the proper development of the drone microbiota. Determining if these bacteria play a role in drone fecundity and health should be a goal of future research efforts.

Importance: Over the last decade, annual honey bee colony loss has increased, resulting in a critical need to determine what factors contribute to honey bee and colony health. Gut microbes have been shown to play important roles in the health of the nonreproductive female honey bee workers, which make up 90% or more of a honey bee colony. However, we currently know very little about the impact of microbes on the health of male honey bees (drones), who only make up a small portion of the colony population but play a very key role in the success of future colonies by mating with virgin queens. Here, we discovered microbes within the reproductive organs of drones and illustrated that social interactions with worker bees are necessary for the proper development of the gut and reproductive tissue microbial communities in drones. Further studies are needed to determine if microbes play an important role in honey bee reproductive health and fitness.