Biology subjects

Ashare, A.

Publications and source records attributed to Ashare, A..

13 recordsLinked to original sources

bioRxiv2026

Molecular-level analysis of serum IgG repertoires in COVID-19-vaccinated people with cystic fibrosis identifies abundant convergent antibodies

BackgroundPeople with cystic fibrosis (pwCF) are at increased risk of severe disease following respiratory viral infections including influenza and respiratory syncytial virus, and were therefore given priority for COVID-19 vaccination. Retrospective epidemiological data have revealed a lower incidence of SARS-CoV-2 infection and a reduced fatality rate among pwCF than in the general population, possibly from the stringent infection control measures and early adoption of protective behaviors. Despite several reports of adequate binding and neutralizing titers after vaccination, the molecular features of vaccine-elicited serum antibody repertoires in pwCF remain unknown. MethodsWe performed high-resolution proteomic analysis of serum IgG, combined with next-generation sequencing of B cells, to quantitatively profile Spike (S)-reactive serological repertoires from nine infection-naive pwCF after two doses of COVID-19 mRNA vaccine. We recombinantly expressed 20 IgG clonotypes from 6 pwCF as monoclonal antibodies (mAbs) and measured their binding and neutralization against vaccine-strain and variant viruses. ResultsAll donors mounted strong binding and neutralizing titers to vaccine-strain virus. Ig-Seq revealed diverse serum repertoires in all vaccinees, with antibodies targeting the receptor-binding domain (RBD) comprising 64% of each circulating repertoire by abundance. Several serum IgG clonotypes identified across our cohort shared identical or similar IGHV and CDRH3 amino acid sequences with serum IgG from other pwCF or S-reactive mAbs isolated from non-CF individuals. These convergent antibodies made up 14.6% of the anti-S repertoires in our cohort, reaching 24.8% in one pwCF. Convergent antibodies tended to be RBD-reactive and enriched in specific IGHV; surprisingly, a higher abundance of convergent antibodies in serum correlated with a lower breadth of serum neutralization. All 20 mAbs bound Wuhan S with high affinity (EC50 < 10 nM), and only RBD-reactive mAbs conferred neutralization to vaccine or variant pseudovirus. While most mAbs bound both the B.1.617.2 (Delta, 17/20) and B.1.1.529 (Omicron, 12/20) strains, convergent mAbs were less likely to bind either variant. ConclusionsPost-vaccine serum IgG repertoires in pwCF are dominated by RBD-focused, high-affinity antibodies and include a substantial convergent component shared with non-CF vaccinees. These findings demonstrate that pwCF mount antibody responses comparable to the general population, and a large group of convergent antibodies may contribute to strain-specific rather than cross-variant immunity.

immunology↗

bioRxiv2026

Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial cells

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a pulmotype detected in [~]34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF. IMPORTANCECystic fibrosis (CF) is a genetic disease characterized by chronic polymicrobial lung infections that, if untreated, are one of the primary causes of death in CF. Elexacaftor/Tezacaftor/Ivacaftor (ETI) has many positive clinical outcomes, but it does not eliminate chronic polymicrobial lung infections or inflammation. Using a new biologically relevant co-culture model, we have demonstrated that bacteria secrete vesicles (bEVs) that contain proteins and RNAs. We observed that these RNA-loaded bEVs are predicted to promote the pathogenesis of chronic CF lung infections by enhancing bacterial virulence and biofilm formation, as well as upregulating the pro-inflammatory response in lung cells. ETI does not ameliorate the response of lung cells to bEVs. Our research will facilitate the development of more effective approaches to eliminate infection and inflammation in CF and other lung diseases characterized by chronic polymicrobial infections and excessive inflammation.

microbiology↗

bioRxiv2026

Within-host population structure, migration, and parallel adaptive evolution of Pseudomonas aeruginosa in cystic fibrosis lung disease

Pseudomonas aeruginosa infections in adults with cystic fibrosis (CF) are comprised of heterogeneous populations, most often tracing ancestry back to a single recent common ancestor. What is not clear is the physical spatial structure within the lung infection population, its stability over time and whether this physical structure leads to different evolutionary trajectories in different adaptive environments. To compare the P. aeruginosa populations across a single lung, we performed whole genome sequence analyses of 450 isolates recovered from lavage samples of the three different lobes of the right lung from a person with mild-to-moderate CF lung disease at three time points over the course of [~]1.5 years. We found that isolates fell into five distinct phylogenetic lineages with evidence for repeated translocation of isolates from different lineages across lobes and loss-of-function mutations in lasR and mucA were present in all 450 isolates. The well-resolved phylogenetic analyses revealed a structured population in which we find the coexistence of a slowly evolving lineage and more rapidly evolving lineages. There is also support for numerous migration events. Further, strong evidence for parallel adaptive mutations in multiple genes revealed distinct evolutionary paths affecting mucoid phenotypes and genetic variation in antibiotic resistance-associated pathways across coexisting populations within a single individual over time. These results provide an example of within-host evolution leading to microheterogeneity that may be useful to consider in future study of infection metapopulations dynamics over the course of chronic infection. IMPORTANCEIndividuals with cystic fibrosis (CF) commonly have chronic lung infections that contain clonally derived Pseudomonas aeruginosa populations with genotypic and phenotypic diversity. This study describes a substantial dataset containing 450 isolates from different lobes of the right lung across three timepoints from an individual with mild-to-moderate CF lung disease. Some regional enrichment for specific lineages with parallel mutations among individual lobes of the lung was observed, but longitudinal analysis also demonstrated that compartmentalization is not strictly maintained and that isolates migrate between lobes of the lung over time. Perspectives on within lung evolution will be important for understanding the pathogen populations in chronic respiratory infections in CF and other diseases.

microbiology↗

bioRxiv2025

Pseudomonas aeruginosa lasR Mutants Resist Phagocytosis and Alter Inflammatory Cytokine Production by Cystic Fibrosis Macrophages

Cystic Fibrosis is characterized by chronic muco-obstructive lung disease and infection. People with CF (pwCF) are often colonized with Pseudomonas aeruginosa for years to decades, allowing for evolutionary adaptation. In chronic P. aeruginosa lung isolates from pwCF, the quorum sensing regulator LasR frequently is nonfunctional, however the factors enabling lasR loss-of-function (LOF) mutant selection are incompletely understood. We hypothesized that LOF mutations in lasR could allow P. aeruginosa to resist the selective pressure of phagocytosis. We found that in multiple strain backgrounds, LasR LOF decreased phagocytosis by both model THP-1 and primary monocyte-derived macrophages. While exogenous administration of the quorum-sensing autoinducer 3-oxo-C12-homoserine-lactone (3OC12HSL) that is made by an enzyme regulated by LasR activity inhibited phagocytosis and mitochondrial respiration, the phagocytosis resistance seen with lasR mutants appears to be bacterial cell intrinsic rather than due to secreted factors. Finally, we found that lasR LOF mutations altered the inflammatory profile upon infection of CF macrophages, with a shift from IL-1 family cytokine expression towards canonical inflammatory markers including IL-6 and TNF. Collectively these data provide a potential explanation for both the prevalence of lasR mutants in the CF lung as well as their association with worse outcomes. ImportanceCystic Fibrosis (CF) is a genetically inherited disease that leads to chronic lung infections. Pseudomonas aeruginosa is often implicated with worsening of lung disease, and it evolves in the lung over time to resist eradication. One of the most commonly disrupted gene targets identified in Pseudomonas aeruginosa isolates from chronically infected CF lungs is lasR, which encodes a transcription factor which regulates multiple virulence factors. What contributes to the apparent fitness of lasR mutants in the CF lung is not well known. Our study shows that lasR loss-of-function (LOF) mutants resist phagocytosis by macrophages, one of the fundamental mechanisms of clearance by the immune system. We identify mechanisms promoting resistance to phagocytosis, and explore the downstream consequences on inflammatory responses. Understanding why lasR mutations arise could inform strategies to eradicate them from the CF lung and improve outcomes.

microbiology↗

bioRxiv2024

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthyThe experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa, which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstractThe anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. (B) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa. 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/578737v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@d9d5e7org.highwire.dtl.DTLVardef@16a5715org.highwire.dtl.DTLVardef@7b35ddorg.highwire.dtl.DTLVardef@10d4dc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that [~]50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower ([~]8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individuals total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

immunology↗

bioRxiv2023

Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung

The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora (Candida) lusitaniae. Whole genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function of Mrs4, a mitochondrial iron transporter. RNA Seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron was much higher in strains with Mrs4 loss of function variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatiditis infection also had a non-synonymous loss of function mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi perhaps for the purposes of adaptation to an iron restricted environment with chronic infections.

microbiology↗

bioRxiv2022

ScRNA-seq Expression of APOC2 and IFI27 Identifies Four Families of Alveolar Macrophage Superclusters in Cystic Fibrosis and Healthy BALF

Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs, interstitial macrophages (IMs), and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified FOLR2+SELENOP+ and SPP1+PLA2G7+ IMs, monocyte subtypes, and dendritic cell 1 (DC1), DC2, migDCs, plasmacytoid DCs, lymphocytes, epithelial cells, and four AM superclusters (families) based on the expression of IFI27 and APOC2 genes. These 4 AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, Cholesterol, Chemokine, Metallothionein, Interferon and small-cluster AMs. Interestingly, the Chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Lastly, other AM datasets were projected onto our dataset. Similar AM superclusters and functional subclusters were observed, along with changes in AM subclusters in individuals infected with COVID-19. Overall, functional specializations of the AM subclusters suggest that there are highly regulated AM niches with defined programming states, highlighting a clear division of labor. Summary BlurbThere are at least 14 AM subtypes; their frequency, along with other immune cells, are highly conserved across individuals suggesting a specific niche exists for each leukocyte population. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/478325v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b9fd85org.highwire.dtl.DTLVardef@b33a9forg.highwire.dtl.DTLVardef@cddcaeorg.highwire.dtl.DTLVardef@1535215_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Metabolic basis for the evolution of a common pathogenic Pseudomonas aeruginosa variant

Microbes frequently evolve in reproducible ways. Here, we show that differences in specific metabolic regulation explain the frequent presence of lasR loss-of-function mutations in the bacterial pathogen Pseudomonas aeruginosa. While LasR contributes to virulence, lasR mutants have been associated with more severe disease. A model based on the intrinsic growth kinetics for a wild type strain and its LasR- derivative, in combination with an experimental evolution based genetic screen and further genetics analyses, indicated that differences in metabolism were sufficient to explain the rise of these common mutant types. The evolution of LasR- lineages in laboratory and clinical isolates depended on activity of the two-component system CbrAB, which modulates substrate prioritization through the catabolite repression control pathway. LasR- lineages frequently arise in cystic fibrosis lung infections and their detection correlates with disease severity. Our analysis of bronchoalveolar lavage fluid metabolomes identified compounds that negatively correlate with lung function, and we show that these compounds support enhanced growth of LasR- cells in a CbrB-controlled manner. We propose that in vivo metabolomes are a major driver of pathogen evolution, which may influence the progression of disease and its treatment.

microbiology↗

bioRxiv2021

Tobramycin suppresses cystic fibrosis lung inflammation by increasing 5' tRNA-fMet halves secreted by P. aeruginosa

Although inhaled tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of P. aeruginosa in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is unclear. Here, we demonstrate that tobramycin increases the abundance of two 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by P. aeruginosa and that the 5' tRNA-fMet halves reduce IL-8 secretion by CF bronchial epithelial cells (CF-HBECs). In mouse lung, the 5' tRNA-fMet halves attenuate KC secretion and neutrophil recruitment. We also report that the 5' tRNA-fMet halves suppress pro-inflammatory network gene expression by an Argonaut 2 (AGO2)-mediated gene silencing mechanism, thereby reducing IL-8 secretion in CF-HBECs. Moreover, tobramycin reduces the IL-8 concentration and neutrophil content in bronchoalveolar lavage fluid of pwCF. Thus, we conclude that tobramycin improves lung function in part by reducing chronic inflammation and neutrophil-mediated lung damage in pwCF.

immunology↗

bioRxiv2021

Aspergillus fumigatus In-Host HOG pathway mutation for Cystic Fibrosis Lung Microenvironment Persistence

The prevalence of Aspergillus fumigatus colonization in individuals with Cystic Fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized. However, there is no consensus for clinical management of A. fumigatus in CF individuals, due largely to uncertainty surrounding A. fumigatus CF pathogenesis and virulence mechanisms. To address this gap in knowledge, a longitudinal series of A. fumigatus isolates from an individual with CF were collected over 4.5 years. Isolate genotypes were defined with whole genome sequencing that revealed both transitory and persistent A. fumigatus in the lung. Persistent lineage isolates grew most readily in a low oxygen culture environment and conidia were more sensitive to oxidative stress inducing conditions compared to non-persistent isolates. Closely related persistent isolates harbor a unique allele of the high osmolarity glycerol (HOG) pathway mitogen activated protein kinase kinase, Pbs2 (pbs2C2). Data suggest this novel pbs2C2 allele arose in vivo and is necessary for the fungal response to osmotic stress in a low oxygen environment through hyperactivation of the HOG (SakA) signaling pathway. Hyperactivation of the HOG pathway through pbs2C2 comes at the cost of decreased conidia stress resistance in the presence of atmospheric oxygen levels. These novel findings shed light on pathoadaptive mechanisms of A. fumigatus in CF, lay the foundation for identifying persistent A. fumigatus isolates that may require antifungal therapy, and highlight considerations for successful culture of persistent fungal CF isolates. ImportanceAspergillus fumigatus infection causes a spectrum of clinical manifestations. For individuals with Cystic Fibrosis (CF), Allergic Bronchopulmonary Aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatus airway persistence in CF disease progression. There currently is little consensus for clinical management of A. fumigatus long-term culture positivity in CF. A better understanding of A. fumigatus pathogenesis mechanisms in CF is expected to yield insights into when antifungal therapies are warranted. Here, a 4.5-year longitudinal collection of A. fumigatus isolates identified a persistent lineage that harbors a unique allele of the Pbs2 MAPKK necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased CF lung fitness at a cost of reduced in vitro growth in standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.

microbiology↗

bioRxiv2020

Balancing positive and negative selection: in vivo evolution of Candida lusitaniae MRR1

The evolution of pathogens in response to selective pressures present during chronic infections can influence persistence, virulence, and the outcomes of antimicrobial therapy. Because subpopulations within an infection can be spatially separated and the host environment can fluctuate, an appreciation of the pathways under selection may be most easily revealed through the analysis of numerous isolates from single infections. Here, we continued our analysis of a set of clonally-derived Clavispora (Candida) lusitaniae isolates from a single chronic lung infection with a striking enrichment in the number of alleles of MRR1. Genetic and genomic analyses found evidence for repeated acquisition of gain-of-function mutations that conferred constitutive Mrr1 activity. In the same population, there were multiple alleles with both gain-of-function mutations and secondary suppressor mutations that either attenuated or abolished the constitutive activity suggesting the presence of counteracting selective pressures. Our studies demonstrated tradeoffs between high Mrr1 activity, which confers resistance to the antifungal fluconazole, host factors, and bacterial products through its regulation of MDR1, and resistance to hydrogen peroxide, a reactive oxygen species produced in the neutrophilic environment associated with this infection. This inverse correlation between high Mrr1 activity and hydrogen peroxide resistance was observed in multiple Candida species and in serial analysis of populations from this individual collected over three years. These data lead us to propose that dynamic or variable selective pressures can be reflected in population genomics and that these dynamics can complicate the drug resistance profile of the population. ImportanceUnderstanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1 alleles in isolates from a single Clavispora (Candida) lusitaniae infection revealed the selection for both high and low Mrr1 activity. Our studies reveal tradeoffs between high Mrr1 activity, which confers resistance to the commonly used antifungal fluconazole, host antimicrobial peptides and bacterial products, and resistance to hydrogen peroxide. This work suggests that spatial or temporal differences within chronic infections can support a large amount of dynamic and parallel evolution, and that Mrr1 activity is under both positive and negative selective pressure to balance different traits that are important for microbial survival.

microbiology↗

bioRxiv2018

Profiling of bacterial and fungal microbial communities in cystic fibrosis sputum using RNA

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect forty two bacterial and fungal genera or species common in cystic fibrosis (CF) sputum, and demonstrated taxon-specificity of these probes as well as a linear response over more than three logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. The relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and, with further development, may provide quick and easily-interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.\n\nImportanceHere we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

microbiology↗