Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung.

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.

Genome rearrangements induce biofilm formation in Escherichia coli C - an old model organism with a new application in biofilm research.

BACKGROUND: Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. RESULTS: Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the - 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli. CONCLUSIONS: Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.

Activation and alliance of regulatory pathways in C. albicans during mammalian infection.

Gene expression dynamics have provided foundational insight into almost all biological processes. Here, we analyze expression of environmentally responsive genes and transcription factor genes to infer signals and pathways that drive pathogen gene regulation during invasive Candida albicans infection of a mammalian host. Environmentally responsive gene expression shows that there are early and late phases of infection. The early phase includes induction of zinc and iron limitation genes, genes that respond to transcription factor Rim101, and genes characteristic of invasive hyphal cells. The late phase includes responses related to phagocytosis by macrophages. Transcription factor gene expression also reflects early and late phases. Transcription factor genes that are required for virulence or proliferation in vivo are enriched among highly expressed transcription factor genes. Mutants defective in six transcription factor genes, three previously studied in detail (Rim101, Efg1, Zap1) and three less extensively studied (Rob1, Rpn4, Sut1), are profiled during infection. Most of these mutants have distinct gene expression profiles during infection as compared to in vitro growth. Infection profiles suggest that Sut1 acts in the same pathway as Zap1, and we verify that functional relationship with the finding that overexpression of either ZAP1 or the Zap1-dependent zinc transporter gene ZRT2 restores pathogenicity to a sut1 mutant. Perturbation with the cell wall inhibitor caspofungin also has distinct gene expression impact in vivo and in vitro. Unexpectedly, caspofungin induces many of the same genes that are repressed early during infection, a phenomenon that we suggest may contribute to drug efficacy. The pathogen response circuitry is tailored uniquely during infection, with many relevant regulatory relationships that are not evident during growth in vitro. Our findings support the principle that virulence is a property that is manifested only in the distinct environment in which host-pathogen interaction occurs.

A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.

In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.

Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol.

Background The Borreliaceae family includes many obligate parasitic bacterial species which are etiologically associated with a myriad of zoonotic borrelioses including Lyme disease and vector-borne relapsing fevers. Infections by the Borreliaceae are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnoses center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration of the causative spirochaetes and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the highly extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity as single target assays cannot provide universal coverage. This within-species heterogeneity is partly due to differences in replicon repertoires and genomic structures that have likely arisen to support the complex Borreliaceae lifecycle in which these parasites have to survive in multiple hosts each with unique immune responses. Results We constructed a Borreliaceae family-level pangenome and characterized the phylogenetic relationships among the constituent taxa which supports the recent taxonomy of splitting the family into at least two genera. Gene content profiles were created for the majority of the Borreliaceae replicons, providing for the first time their unambiguous molecular typing. Conclusion Our characterization of the Borreliaceae pan-genome supports the splitting of the former Borrelia genus into two genera and provides for the phylogenetic placement of several non-species designated isolates. Mining this family-level pangenome will enable precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions.

Genetic Adaptation and Acquisition of Macrolide Resistance in Haemophilus spp. during Persistent Respiratory Tract Colonization in Chronic Obstructive Pulmonary Disease (COPD) Patients Receiving Long-Term Azithromycin Treatment.

Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain CU1000N.

Here, we report the complete genome sequence of Aggregatibacter actinomycetemcomitans strain CU1000N. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.

Local Adaptation and the Evolution of Genome Architecture in Threespine Stickleback.

Theory predicts that local adaptation should favor the evolution of a concentrated genetic architecture, where the alleles driving adaptive divergence are tightly clustered on chromosomes. Adaptation to marine versus freshwater environments in threespine stickleback has resulted in an architecture that seems consistent with this prediction: divergence among populations is mainly driven by a few genomic regions harboring multiple quantitative trait loci for environmentally adapted traits, as well as candidate genes with well-established phenotypic effects. One theory for the evolution of these "genomic islands" is that rearrangements remodel the genome to bring causal loci into tight proximity, but this has not been studied explicitly. We tested this theory using synteny analysis to identify micro- and macro-rearrangements in the stickleback genome and assess their potential involvement in the evolution of genomic islands. To identify rearrangements, we conducted a de novo assembly of the closely related tubesnout (Aulorhyncus flavidus) genome and compared this to the genomes of threespine stickleback and two other closely related species. We found that small rearrangements, within-chromosome duplications, and lineage-specific genes (LSGs) were enriched around genomic islands, and that all three chromosomes harboring large genomic islands have experienced macro-rearrangements. We also found that duplicates and micro-rearrangements are 9.9× and 2.9× more likely to involve genes differentially expressed between marine and freshwater genotypes. While not conclusive, these results are consistent with the explanation that strong divergent selection on candidate genes drove the recruitment of rearrangements to yield clusters of locally adaptive loci.

Transition of Serotype 35B Pneumococci From Commensal to Prevalent Virulent Strain in Children.

In our community-based prospective cohort study in young children, we observed a significant increase in pneumococcal serotype 35B nasopharyngeal (NP) commensal colonization during the 2011-2014 timeframe, but these strains were not associated with disease. Beginning in 2015 and continuing through to the present, the serotype 35B virulence changed, and it became the dominant bacteria isolated and associated with pneumococcal acute otitis-media (AOM) in our cohort. We performed comparative analyses of 250 35B isolates obtained from 140 children collected between 2006 and 2019. Changes in prevalence, clonal-complex composition, and antibiotic resistance were analyzed. Seventy-two (29%) of 35B isolates underwent whole-genome sequencing to investigate genomic changes associated with the shift in virulence that resulted in increased rates of 35B-associated AOM disease. 35B strains that were commensals and AOM disease-causing were mainly associated with sequence type (ST) 558. Antibiotic concentrations of ß-lactams and ofloxacin necessary to inhibit growth of 35B strains rose significantly (2006-2019) (p<0.005). However, only isolates from the 35B/ST558 showed significant increases in MIC50 of penicillin and ofloxacin between the years 2006-2014 and 2015-2019 (p=0.007 and p<0.0001). One hundred thirty-eight SNPs located in 34 different genes were significantly associated with post-2015 strains. SNPs were found in nrdG (metal binding, 10%); metP and metN (ABC transporter, 9%); corA (Mg2+ transporter, 6%); priA (DNA replication, 5%); and on the enzymic gene ldcB (LD-carboxypeptidase, 3%). Pneumococcal serotype 35B strains was a common NP commensal during 2010-2014. In 2015, a shift in increasing number of AOM cases occurred in young children caused by 35B, that was associated with changes in genetic composition and antibiotic susceptibility.

Genome-wide analysis of DNA uptake across the outer membrane of naturally competent Haemophilus influenzae.

The genomes of naturally competent Pasteurellaceae and Neisseriaceae have many short uptake sequences (USS), which allow them to distinguish self-DNA from foreign DNA. To fully characterize this preference we developed genome-wide maps of DNA uptake using both a sequence-based computational model and genomic DNA that had been sequenced after uptake by and recovery from competent Haemophilus influenzae cells. When DNA fragments were shorter than the average USS spacing of ∼1,000 bp, sharp peaks of uptake were centered at USS and separated by valleys with 1000-fold lower uptake. Long DNA fragments (1.5-17 kb) gave much less variation, with 90% of positions having uptake within 2-fold of the mean. All detectable uptake biases arose from sequences that fit the USS uptake motif. Simulated competition predicted that, in its respiratory tract environment, H. influenzae will efficiently take up its own DNA even when human DNA is present in 100-fold excess.

Whole genome sequencing of Streptomyces actuosus ISP-5337, Streptomyces sioyaensis B-5408, and Actinospica acidiphila B-2296 reveals secondary metabolomes with antibiotic potential.

Streptomycetes are bacteria of biotechnological importance since they are avid producers of secondary metabolites, including antibiotics. Progress in genome mining has recently shown that Streptomyces species encode for many biosynthetic gene clusters which are mostly unexplored. Here, we selected three Actinomycetes species for whole genome sequencing that are known to produce potent thiopeptide antibiotics. Streptomyces actuosus biosynthesizes nosiheptide, Streptomyces sioyaensis produces siomycin, and Actinospica acidiphila is a member of the Actinomycete subfamily. Bioinformatic analyses demonstrated diverse secondary metabolomes with multiple antibiotic-encoding gene clusters. Detailed mass spectrometry analysis of metabolite extracts verified the active expression of nosiheptide and siomycin from S. actuosus and S. sioyaensis while fractionation of the bacterial extracts and subsequent challenge against Staphylococcus aureus demonstrated potent antibiotic activity of fractions containing these compounds. Whole genome sequencing of these species facilitates future bioengineering efforts for thiopeptides and characterization of relevant secondary metabolites.

A competence-regulated toxin-antitoxin system in Haemophilus influenzae.

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ('toxTA'), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy- and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes.

BACKGROUND: Pan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences' (PacBio's) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data. RESULTS: Analysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of > 90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site. CONCLUSIONS: Our microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt ) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.

Antagonistic Pleiotropy in the Bifunctional Surface Protein FadL (OmpP1) during Adaptation of Haemophilus influenzae to Chronic Lung Infection Associated with Chronic Obstructive Pulmonary Disease.

Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9 years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL's interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi's ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium's ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts ΔfadL strains' niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways.

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain IDH781.

We report here the complete genomic sequence and methylome of Aggregatibacter actinomycetemcomitans strain IDH781. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.