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 pro les 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.

Mitochondrially targeted proximity biotinylation and proteomic analysis in Plasmodium falciparum.

Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.

Pre-vaccination frequency of circulatory Tfh is associated with robust immune response to TV003 dengue vaccine.

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees' ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.

The bacterial microbiota of Hunner lesion interstitial cystitis/bladder pain syndrome.

OBJECTIVE: To undertake the first comprehensive evaluation of the urinary microbiota associated with Hunner lesion (HL) interstitial cystitis/bladder pain syndrome (IC/BPS). Despite no previous identification of a distinct IC/BPS microbial urotype, HL IC/BPS, an inflammatory subtype of IC/BPS, was hypothesized most likely to be associated with a specific bacterial species or microbial pattern. PARTICIPANTS AND METHODS: The bacterial microbiota of midstream urine specimens from HL IC/BPS and age- and gender-matched IC/BPS patients without HL (non-HL IC/BPS) were examined using the pan-bacterial domain clinical-level molecular diagnostic Pacific Biosciences full-length 16S gene sequencing protocol, informatics pipeline and database. We characterized the differential presence, abundances, and diversity of species, as well as gender-specific differences between and among HL and non-HL IC/BPS patients. RESULTS: A total of 59 patients with IC/BPS were enrolled (29 HL, 30 non-HL; 43 women, 16 men) from a single centre and the microbiota in midstream urine specimens was available for comparison. The species abundance differentiation between the HL and non-HL groups (12 species) was not significantly different after Bonferroni adjustments for multiple comparisons. Similarly, the nine differentiating species noted between female HL and non-HL patients were not significantly different after similar statistical correction. However, four species abundances (out of the 10 species differences identified prior to correction) remained significantly different between male HL and non-HL subjects: Negativicoccus succinivorans, Porphyromonas somerae, Mobiluncus curtisii and Corynebacterium renale. Shannon diversity metrics showed significantly higher diversity among HL male patients than HL female patients (P = 0.045), but no significant diversity differences between HL and non-HL patients overall. CONCLUSIONS: We were not able to identify a unique pathogenic urinary microbiota that differentiates all HL from all non-HL IC/BPS. It is likely that the male-specific differences resulted from colonization/contamination remote from the bladder. We were not able to show that bacteria play an important role in patients with HL IC/BPS.

Phase Variation in HMW1A Controls a Phenotypic Switch in Haemophilus influenzae Associated with Pathoadaptation during Persistent Infection.

Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth.

Panel 3: Genomics, precision medicine and targeted therapies.

OBJECTIVE: To review the most recent advances in human and bacterial genomics as applied to pathogenesis and clinical management of otitis media. DATA SOURCES: PubMed articles published since the last meeting in June 2015 up to June 2019. REVIEW METHODS: A panel of experts in human and bacterial genomics of otitis media was formed. Each panel member reviewed the literature in their respective fields and wrote draft reviews. The reviews were shared with all panel members, and a merged draft was created. The panel met at the 20th International Symposium on Recent Advances in Otitis Media in June 2019, discussed the review and refined the content. A final draft was made, circulated, and approved by the panel members. CONCLUSION: Trans-disciplinary approaches applying pan-omic technologies to identify human susceptibility to otitis media and to understand microbial population dynamics, patho-adaptation and virulence mechanisms are crucial to the development of novel, personalized therapeutics and prevention strategies for otitis media. IMPLICATIONS FOR PRACTICE: In the future otitis media prevention strategies may be augmented by mucosal immunization, combination vaccines targeting multiple pathogens, and modulation of the middle ear microbiome. Both treatment and vaccination may be tailored to an individual's otitis media phenotype as defined by molecular profiles obtained by using rapidly developing techniques in microbial and host genomics.

Author Correction: Interaction between the microbiome and TP53 in human lung cancer.

Following publication of the original paper [1], the authors submitted a new Additional file 5 to replace the one containing formatting issues. The updated Additional file 5 is published in this correction.

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.

Interaction between the microbiome and TP53 in human lung cancer.

BACKGROUND: Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort. RESULTS: Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas. CONCLUSIONS: The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression.

Globally, a chronic hepatitis B virus (HBV) infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication.

Seventeen Sxy-dependent cyclic AMP receptor protein site-regulated genes are needed for natural transformation in Haemophilus influenzae.

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized.

Natural transformation of Gallibacterium anatis.

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.

Superhelical duplex destabilization and the recombination position effect.

The susceptibility to recombination of a plasmid inserted into a chromosome varies with its genomic position. This recombination position effect is known to correlate with the average G+C content of the flanking sequences. Here we propose that this effect could be mediated by changes in the susceptibility to superhelical duplex destabilization that would occur. We use standard nonparametric statistical tests, regression analysis and principal component analysis to identify statistically significant differences in the destabilization profiles calculated for the plasmid in different contexts, and correlate the results with their measured recombination rates. We show that the flanking sequences significantly affect the free energy of denaturation at specific sites interior to the plasmid. These changes correlate well with experimentally measured variations of the recombination rates within the plasmid. This correlation of recombination rate with superhelical destabilization properties of the inserted plasmid DNA is stronger than that with average G+C content of the flanking sequences. This model suggests a possible mechanism by which flanking sequence base composition, which is not itself a context-dependent attribute, can affect recombination rates at positions within the plasmid.

Genome-wide mapping and assembly of structural variant breakpoints in the mouse genome.

Structural variation (SV) is a rich source of genetic diversity in mammals, but due to the challenges associated with mapping SV in complex genomes, basic questions regarding their genomic distribution and mechanistic origins remain unanswered. We have developed an algorithm (HYDRA) to localize SV breakpoints by paired-end mapping, and a general approach for the genome-wide assembly and interpretation of breakpoint sequences. We applied these methods to two inbred mouse strains: C57BL/6J and DBA/2J. We demonstrate that HYDRA accurately maps diverse classes of SV, including those involving repetitive elements such as transposons and segmental duplications; however, our analysis of the C57BL/6J reference strain shows that incomplete reference genome assemblies are a major source of noise. We report 7196 SVs between the two strains, more than two-thirds of which are due to transposon insertions. Of the remainder, 59% are deletions (relative to the reference), 26% are insertions of unlinked DNA, 9% are tandem duplications, and 6% are inversions. To investigate the origins of SV, we characterized 3316 breakpoint sequences at single-nucleotide resolution. We find that approximately 16% of non-transposon SVs have complex breakpoint patterns consistent with template switching during DNA replication or repair, and that this process appears to preferentially generate certain classes of complex variants. Moreover, we find that SVs are significantly enriched in regions of segmental duplication, but that this effect is largely independent of DNA sequence homology and thus cannot be explained by non-allelic homologous recombination (NAHR) alone. This result suggests that the genetic instability of such regions is often the cause rather than the consequence of duplicated genomic architecture.