Novel PHEX gene locus-specific database: Comprehensive characterization of vast number of variants associated with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemia, is caused by disrupting variants in the PHEX gene, located on the X chromosome. XLH is inherited in an X-linked pattern with complete penetrance observed for both males and females. Patients experience lifelong symptoms resulting from chronic hypophosphatemia, including impaired bone mineralization, skeletal deformities, growth retardation, and diminished quality of life. This chronic condition requires life-long management with disease-specific therapies, which can improve patient outcomes especially when initiated early in life. To centralize and disseminate PHEX variant information, we have established a new PHEX gene locus-specific database, PHEX LSDB. As of April 30, 2021, 870 unique PHEX variants, compiled from an older database of PHEX variants, a comprehensive literature search, a sponsored genetic testing program, and XLH clinical trials, are represented in the PHEX LSDB. This resource is publicly available on an interactive, searchable website (https://www.rarediseasegenes.com/), which includes a table of variants and associated data, graphical/tabular outputs of genotype-phenotype analyses, and an online submission form for reporting new PHEX variants. The database will be updated regularly with new variants submitted on the website, identified in the published literature, or shared from genetic testing programs.

Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis.

BACKGROUND: The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I. ovatus Ehrlichia (IOE) agent], causes acute fatal infection in laboratory mice that resembles acute fatal human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. As there is no small laboratory animal model to study fatal human ehrlichiosis, Ehrlichia sp. HF provides a needed disease model. However, the inability to culture Ehrlichia sp. HF and the lack of genomic information have been a barrier to advance this animal model. In addition, Ehrlichia sp. HF has several designations in the literature as it lacks a taxonomically recognized name. RESULTS: We stably cultured Ehrlichia sp. HF in canine histiocytic leukemia DH82 cells from the HF strain-infected mice, and determined its complete genome sequence. Ehrlichia sp. HF has a single double-stranded circular chromosome of 1,148,904 bp, which encodes 866 proteins with a similar metabolic potential as E. chaffeensis. Ehrlichia sp. HF encodes homologs of all virulence factors identified in E. chaffeensis, including 23 paralogs of P28/OMP-1 family outer membrane proteins, type IV secretion system apparatus and effector proteins, two-component systems, ankyrin-repeat proteins, and tandem repeat proteins. Ehrlichia sp. HF is a novel species in the genus Ehrlichia, as demonstrated through whole genome comparisons with six representative Ehrlichia species, subspecies, and strains, using average nucleotide identity, digital DNA-DNA hybridization, and core genome alignment sequence identity. CONCLUSIONS: The genome of Ehrlichia sp. HF encodes all known virulence factors found in E. chaffeensis, substantiating it as a model Ehrlichia species to study fatal human ehrlichiosis. Comparisons between Ehrlichia sp. HF and E. chaffeensis will enable identification of in vivo virulence factors that are related to host specificity, disease severity, and host inflammatory responses. We propose to name Ehrlichia sp. HF as Ehrlichia japonica sp. nov. (type strain HF), to denote the geographic region where this bacterium was initially isolated.

Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient's lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Signature of adaptive evolution in olfactory receptor genes in Cory's Shearwater supports molecular basis for smell in procellariiform seabirds.

Olfactory receptors (ORs), encoded by the largest vertebrate multigene family, enable the detection of thousands of unique odorants in the environment and consequently play a critical role in species survival. Here, we advance our knowledge of OR gene evolution in procellariiform seabirds, an avian group which relies on the sense of olfaction for critical ecological functions. We built a cosmid library of Cory's Shearwater (Calonectris borealis) genomic DNA, a model species for the study of olfaction-based navigation, and sequence OR gene-positive cosmid clones with a combination of sequencing technologies. We identified 220 OR open reading frames, 20 of which are full length, intact OR genes, and found a large ratio of partial and pseudogenes to intact OR genes (2:1), suggestive of a dynamic mode of evolution. Phylogenetic analyses revealed that while a few genes cluster with those of other sauropsid species in a γ (gamma) clade that predates the divergence of different avian lineages, most genes belong to an avian-specific γ-c clade, within which sequences cluster by species, suggesting frequent duplication and/or gene conversion events. We identified evidence of positive selection on full length γ-c clade genes. These patterns are consistent with a key role of adaptation in the functional diversification of olfactory receptor genes in a bird lineage that relies extensively on olfaction.

The Streptococcus agalactiae Stringent Response Enhances Virulence and Persistence in Human Blood.

Streptococcus agalactiae (group B Streptococcus [GBS]) causes serious infections in neonates. We previously reported a transposon sequencing (Tn-seq) system for performing genomewide assessment of gene fitness in GBS. In order to identify molecular mechanisms required for GBS to transition from a mucosal commensal lifestyle to bloodstream invasion, we performed Tn-seq on GBS strain A909 with human whole blood. Our analysis identified 16 genes conditionally essential for GBS survival in blood, of which 75% were members of the capsular polysaccharide (cps) operon. Among the non-cps genes identified as conditionally essential was relA, which encodes an enzyme whose activity is central to the bacterial stringent response-a conserved adaptation to environmental stress. We used blood coincubation studies of targeted knockout strains to confirm the expected growth defects of GBS deficient in capsule or stringent response activation. Unexpectedly, we found that the relA knockout strains demonstrated decreased expression of ß-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Furthermore, chemical activation of the stringent response with serine hydroxamate increased ß-hemolysin/cytolysin expression. To establish a mechanism by which the stringent response leads to increased cytotoxicity, we performed transcriptome sequencing (RNA-seq) on two GBS strains grown under stringent response or control conditions. This revealed a conserved decrease in the expression of genes in the arginine deiminase pathway during stringent response activation. Through coincubation with supplemental arginine and the arginine antagonist canavanine, we show that arginine availability is a determinant of GBS cytotoxicity and that the pathway between stringent response activation and increased virulence is arginine dependent.

Transcriptional Variation of Diverse Enteropathogenic Escherichia coli Isolates under Virulence-Inducing Conditions.

Enteropathogenic Escherichia coli (EPEC) bacteria are a diverse group of pathogens that cause moderate to severe diarrhea in young children in developing countries. EPEC isolates can be further subclassified as typical EPEC (tEPEC) isolates that contain the bundle-forming pilus (BFP) or as atypical EPEC (aEPEC) isolates that do not contain BFP. Comparative genomics studies have recently highlighted the considerable genomic diversity among EPEC isolates. In the current study, we used RNA sequencing (RNA-Seq) to characterize the global transcriptomes of eight tEPEC isolates representing the identified genomic diversity, as well as one aEPEC isolate. The global transcriptomes were determined for the EPEC isolates under conditions of laboratory growth that are known to induce expression of virulence-associated genes. The findings demonstrate that unique genes of EPEC isolates from diverse phylogenomic lineages contribute to variation in their global transcriptomes. There were also phylogroup-specific differences in the global transcriptomes, including genes involved in iron acquisition, which had significant differential expression in the EPEC isolates belonging to phylogroup B2. Also, three EPEC isolates from the same phylogenomic lineage (EPEC8) had greater levels of similarity in their genomic content and exhibited greater similarities in their global transcriptomes than EPEC from other lineages; however, even among closely related isolates there were isolate-specific differences among their transcriptomes. These findings highlight the transcriptional variability that correlates with the previously unappreciated genomic diversity of EPEC. IMPORTANCE Recent studies have demonstrated that there is considerable genomic diversity among EPEC isolates; however, it is unknown if this genomic diversity leads to differences in their global transcription. This study used RNA-Seq to compare the global transcriptomes of EPEC isolates from diverse phylogenomic lineages. We demonstrate that there are lineage- and isolate-specific differences in the transcriptomes of genomically diverse EPEC isolates during growth under in vitro virulence-inducing conditions. This study addressed biological variation among isolates of a single pathovar in an effort to demonstrate that while each of these isolates is considered an EPEC isolate, there is significant transcriptional diversity among members of this pathovar. Future studies should consider whether this previously undescribed transcriptional variation may play a significant role in isolate-specific variability of EPEC clinical presentations.

Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing.

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.

Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli.

Escherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.

Analysis of complete genome sequence and major surface antigens of Neorickettsia helminthoeca, causative agent of salmon poisoning disease.

Neorickettsia helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. We determined and analysed the complete genome sequence of N. helminthoeca: a single small circular chromosome of 884 232 bp encoding 774 potential proteins. N. helminthoeca is unable to synthesize lipopolysaccharides and most amino acids, but is capable of synthesizing vitamins, cofactors, nucleotides and bacterioferritin. N. helminthoeca is, however, distinct from majority of the family Anaplasmataceae to which it belongs, as it encodes nearly all enzymes required for peptidoglycan biosynthesis, suggesting its structural hardiness and inflammatory potential. Using sera from dogs that were experimentally infected by feeding with parasitized fish or naturally infected in southern California, Western blot analysis revealed that among five predicted N. helminthoeca outer membrane proteins, P51 and strain-variable surface antigen were uniformly recognized. Our finding will help understanding pathogenesis, prevalence of N. helminthoeca infection among trematodes, canids and potentially other animals in nature to develop effective SPD diagnostic and preventive measures. Recent progresses in large-scale genome sequencing have been uncovering broad distribution of Neorickettsia spp., the comparative genomics will facilitate understanding of biology and the natural history of these elusive environmental bacteria.

RNAseq Analysis of the Drosophila Response to the Entomopathogenic Nematode Steinernema.

Drosophila melanogaster is an outstanding model to study the molecular and functional basis of host-pathogen interactions. Currently, our knowledge of microbial infections in D. melanogaster is well understood; however, the response of flies to nematode infections is still in its infancy. Here, we have used the potent parasitic nematode Steinernema carpocapsae, which lives in mutualism with its endosymbiotic bacteria Xenorhabdus nematophila, to examine the transcriptomic basis of the interaction between D. melanogaster and entomopathogenic nematodes. We have employed next-generation RNA sequencing (RNAseq) to investigate the transcriptomic profile of D. melanogaster larvae in response to infection by S. carpocapsae symbiotic (carrying X. nematophila) or axenic (lacking X. nematophila) nematodes. Bioinformatic analyses have identified the strong induction of genes that are associated with the peritrophic membrane and the stress response, as well as several genes that participate in developmental processes. We have also found that genes with different biological functions are enriched in D. melanogaster larvae responding to either symbiotic or axenic nematodes. We further show that while symbiotic nematode infection enriched certain known immune-related genes, axenic nematode infection enriched several genes associated with chitin binding, lipid metabolic functions, and neuroactive ligand receptors. In addition, we have identified genes with a potential role in nematode recognition and genes with potential antinematode activity. Findings from this study will undoubtedly set the stage for the identification of key regulators of antinematode immune mechanisms in D. melanogaster, as well as in other insects of socioeconomic importance.

Truncated TrkB.T1-Mediated Astrocyte Dysfunction Contributes to Impaired Motor Function and Neuropathic Pain after Spinal Cord Injury.

Following spinal cord injury (SCI), astrocytes demonstrate long-lasting reactive changes, which are associated with the persistence of neuropathic pain and motor dysfunction. We previously demonstrated that upregulation of trkB.T1, a truncated isoform of the brain-derived neurotrophic factor receptor (BDNF), contributes to gliosis after SCI, but little is known about the effects of trkB.T1 on the function of astrocytes. As trkB.T1 is the sole isoform of trkB receptors expressed on astrocytes, we examined the function of trkB.T1-driven astrocytes in vitro and in vivo Immunohistochemistry showed that trkB.T1+ cells were significantly upregulated 7 d after injury, with sustained elevation in white matter through 8 weeks. The latter increase was predominantly found in astrocytes. TrkB.T1 was also highly expressed by neurons and microglia/macrophages at 7 d after injury and declined by 8 weeks. RNA sequencing of cultured astrocytes derived from trkB.T1+/+ (WT) and trkB.T1-/- (KO) mice revealed downregulation of migration and proliferation pathways in KO astrocytes. KO astrocytes also exhibited slower migration/proliferation in vitro in response to FBS or BDNF compared with WT astrocytes. Reduced proliferation of astrocytes was also confirmed after SCI in astrocyte-specific trkB.T1 KO mice; using mechanical allodynia and pain-related measurements on the CatWalk, these animals also showed reduced hyperpathic responses, along with improved motor coordination. Together, our data indicate that trkB.T1 in astrocytes contributes to neuropathic pain and neurological dysfunction following SCI, suggesting that trkB.T1 may provide a novel therapeutic target for SCI.SIGNIFICANCE STATEMENT Neuropathic pain after spinal cord injury (SCI) may in part be caused by upregulation of the brain-derived neurotrophic factor (BDNF) receptor trkB.T1, a truncated isoform of BDNF. TrkB.T1 is the only isoform of tropomyosin-related receptor kinase type B (trkB) receptors expressed on astrocytes. Here, we showed that trkB.T1 is significantly increased in the injured mouse spinal cord, where it is predominantly found in astrocytes. RNA sequencing of cultured astrocytes demonstrated downregulation of migration and proliferation pathways in trkB.T1 KO astrocytes. This was validated in vivo, where deletion of trkB.T1 in astrocytes reduced cell proliferation and migration. After SCI, astrocyte-specific trkB.T1 KO mice showed reduced hyperpathic responses and improved motor coordination. Therefore, the trkB.T1 receptor plays a significant pathophysiological role after SCI, and may provide a novel therapeutic target for SCI.

Effect of a small molecule Lipid II binder on bacterial cell wall stress.

We have recently identified small molecule compounds that act as binders of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprised a hydrophilic head group that includes a peptidoglycan subunit composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) coupled to a short pentapeptide moiety. This headgroup is coupled to a long bactoprenol chain via a pyrophosphate group. Here, we report on the cell wall activity relationship of dimethyl-3-methyl(phenyl)amino-ethenylcyclohexylidene-propenyl-3-ethyl-1,3-benzothiazolium iodide (compound 5107930) obtained by functional and genetic analyses. Our results indicate that compounds bind to Lipid II and cause specific upregulation of the vancomycin-resistance associated gene vraX. vraX is implicated in the cell wall stress stimulon that confers glycopeptide resistance. Our small molecule Lipid II inhibitor retained activity against strains of Staphylococcus aureus mutated in genes encoding the cell wall stress stimulon. This suggests the feasibility of developing this new scaffold as a therapeutic agent in view of increasing glycopeptide resistance.

IL-17 Receptor Signaling in Oral Epithelial Cells Is Critical for Protection against Oropharyngeal Candidiasis.

Signaling through the IL-17 receptor (IL-17R) is required to prevent oropharyngeal candidiasis (OPC) in mice and humans. However, the IL-17-responsive cell type(s) that mediate protection are unknown. Using radiation chimeras, we were able to rule out a requirement for IL-17RA in the hematopoietic compartment. We saw remarkable concordance of IL-17-controlled gene expression in C. albicans-infected human oral epithelial cells (OECs) and in tongue tissue from mice with OPC. To interrogate the role of the IL-17R in OECs, we generated mice with conditional deletion of IL-17RA in superficial oral and esophageal epithelial cells (Il17raΔK13). Following oral Candida infection, Il17raΔK13 mice exhibited fungal loads and weight loss indistinguishable from Il17ra-/- mice. Susceptibility in Il17raΔK13 mice correlated with expression of the antimicrobial peptide ß-defensin 3 (BD3, Defb3). Consistently, Defb3-/- mice were susceptible to OPC. Thus, OECs dominantly control IL-17R-dependent responses to OPC through regulation of BD3 expression.

Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples.

Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont. While a suitable method is commercially available for mammalian samples of this type, development of such methods has languished for invertebrate samples. Furthermore, a common method across multiple taxa would facilitate comparisons between bacteria in invertebrate vectors and their vertebrate hosts. Here, a method is described to concurrently remove polyadenylated transcripts, prokaryotic rRNA, and eukaryotic rRNA, including those with low amounts of starting material (e.g. 100 ng). In a Wolbachia-Drosophila system, this bacterial mRNA enrichment yielded a 3-fold increase in Wolbachia mRNA abundance and a concomitant 3.3-fold increase in the percentage of transcripts detected. More specifically, 70% of the genome could be recovered by transcriptome sequencing compared to 21% in the total RNA. Sequencing of similar bacterial mRNA-enriched samples generated from Ehrlichia-infected canine cells covers 93% of the Ehrlichia genome, suggesting ubiquitous transcription across the entire Ehrlichia chaffeensis genome. This technique can potentially be used to enrich bacterial mRNA in many studies of host-microbe interactions.

Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and ß-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role.

An integrated genomic and transcriptomic survey of mucormycosis-causing fungi.

Mucormycosis is a life-threatening infection caused by Mucorales fungi. Here we sequence 30 fungal genomes, and perform transcriptomics with three representative Rhizopus and Mucor strains and with human airway epithelial cells during fungal invasion, to reveal key host and fungal determinants contributing to pathogenesis. Analysis of the host transcriptional response to Mucorales reveals platelet-derived growth factor receptor B (PDGFRB) signaling as part of a core response to divergent pathogenic fungi; inhibition of PDGFRB reduces Mucorales-induced damage to host cells. The unique presence of CotH invasins in all invasive Mucorales, and the correlation between CotH gene copy number and clinical prevalence, are consistent with an important role for these proteins in mucormycosis pathogenesis. Our work provides insight into the evolution of this medically and economically important group of fungi, and identifies several molecular pathways that might be exploited as potential therapeutic targets.

Use of Comparative Genomics To Characterize the Diversity of Acinetobacter baumannii Surveillance Isolates in a Health Care Institution.

Despite the increasing prevalence of the nosocomial pathogen Acinetobacter baumannii, little is known about which genomic components contribute to clinical presentation of this important pathogen. Most whole-genome comparisons of A. baumannii have focused on specific genomic regions associated with phenotypes in a limited number of genomes. In this work, we describe the results of a whole-genome comparative analysis of 254 surveillance isolates of Acinetobacter species, 203 of which were A. baumannii, isolated from perianal swabs and sputum samples collected as part of an infection control active surveillance program at the University of Maryland Medical Center. The collection of surveillance isolates includes both carbapenem-susceptible and -resistant isolates. Based on the whole-genome phylogeny, the A. baumannii isolates collected belong to two major phylogenomic lineages. Results from multilocus sequence typing indicated that one of the major phylogenetic groups of A. baumannii was comprised solely of strains from the international clonal lineage 2. The genomic content of the A. baumannii isolates was examined using large-scale BLAST score ratio analysis to identify genes that are associated with carbapenem-susceptible and -resistant isolates, as well as genes potentially associated with the source of isolation. This analysis revealed a number of genes that were exclusive or at greater frequency in each of these classifications. This study is the most comprehensive genomic comparison of Acinetobacter isolates from a surveillance study to date and provides important information that will contribute to our understanding of the success of A. baumannii as a human pathogen.

RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates.

Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis.

The genome sequence of four isolates from the family Lichtheimiaceae.

This study reports the release of draft genome sequences of two isolates of Lichtheimia corymbifera and two isolates of L. ramosa. Phylogenetic analyses indicate that the two L. corymbifera strains (CDC-B2541 and 008-049) are closely related to the previously sequenced L. corymbifera isolate (FSU 9682) while our two L. ramosa strains CDC-B5399 and CDC-B5792 cluster apart from them. These genome sequences will further the understanding of intraspecies and interspecies genetic variation within the Mucoraceae family of pathogenic fungi.