Publications by authors named "Hervé Tettelin"

99 Publications

Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits.

PLoS Pathog 2021 Mar 8;17(3):e1009116. Epub 2021 Mar 8.

Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.
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http://dx.doi.org/10.1371/journal.ppat.1009116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971860PMC
March 2021

Genome-wide association study in patients with pulmonary complex disease.

Eur Respir J 2021 Feb 4. Epub 2021 Feb 4.

Department of Infectious Diseases, Keio University School of Medicine, Tokyo, Japan.

Rationale: Nontuberculous mycobacteria (NTM) are environmental mycobacteria that can cause a chronic progressive lung disease. Although epidemiological data indicate potential genetic predisposition, its nature remains unclear.

Objectives: We aimed to identify host susceptibility loci for complex (MAC), the most common NTM pathogen.

Methods: This genome-wide association study (GWAS) was conducted in Japanese patients with pulmonary MAC and healthy controls, followed by genotyping of candidate single-nucleotide polymorphisms (SNPs) in another Japanese cohort. For verification by Korean and European ancestry, we performed SNP genotyping.

Results: The GWAS discovery set included 475 pulmonary MAC cases and 417 controls. Both GWAS and replication analysis of 591 pulmonary MAC cases and 718 controls revealed the strongest association with chromosome 16p21, particularly with rs109592 (p=1.64E-13, odds ratio=0.54), which is in an intronic region of the calcineurin like EF-hand protein 2 (). Expression quantitative trait loci analysis demonstrated an association with lung CHP2 expression. CHP2 was expressed in the lung tissue in pulmonary MAC disease. This SNP was associated with the nodular bronchiectasis subtype. This SNP was also significantly associated with the disease in patients of Korean (p=2.18E-12, odds ratio=0.54) and European (p=5.12E-03, odds ratio=0.63) ancestry.

Conclusions: We identified rs109592 in the locus as a susceptibility marker for pulmonary MAC disease.
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http://dx.doi.org/10.1183/13993003.02269-2019DOI Listing
February 2021

Sugar-Coated Killer: Serotype 3 Pneumococcal Disease.

Front Cell Infect Microbiol 2020 23;10:613287. Epub 2020 Dec 23.

Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL, United States.

Capsular polysaccharide (CPS), which surrounds the bacteria, is one of the most significant and multifaceted contributors to virulence. Capsule prevents entrapment in mucus during colonization, traps water to protect against desiccation, can serve as an energy reserve, and protects the bacterium against complement-mediated opsonization and immune cell phagocytosis. To date, 100 biochemically and serologically distinct capsule types have been identified for ; 20 to 30 of which have well-defined propensity to cause opportunistic human infection. Among these, serotype 3 is perhaps the most problematic as serotype 3 infections are characterized as having severe clinical manifestations including empyema, bacteremia, cardiotoxicity, and meningitis; consequently, with a fatality rate of 30%-47%. Moreover, serotype 3 resists antibody-mediated clearance despite its inclusion in the current 13-valent conjugate vaccine formulation. This review covers the role of capsule in pneumococcal pathogenesis and the importance of serotype 3 on human disease. We discuss how serotype 3 capsule synthesis and presentation on the bacterial surface is distinct from other serotypes, the biochemical and physiological properties of this capsule type that facilitate its ability to cause disease, and why existing vaccines are unable to confer protection. We conclude with discussion of the clonal properties of serotype 3 and how these have changed since introduction of the 13-valent vaccine in 2000.
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http://dx.doi.org/10.3389/fcimb.2020.613287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786310PMC
December 2020

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

Virulence 2020 12;11(1):1656-1673

Department of Laboratory Medicine, Clinical Center, NIH , Bethesda, USA.

: We characterized 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. : We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct BCG-vaccine derived clinical strains were recovered from the patient's lungs and brain. : 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. : Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 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 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. 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. : 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.
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http://dx.doi.org/10.1080/21505594.2020.1848108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781554PMC
December 2020

An in vivo atlas of host-pathogen transcriptomes during colonization and disease.

Proc Natl Acad Sci U S A 2020 12 14;117(52):33507-33518. Epub 2020 Dec 14.

Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201;

() colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three strains. We identified genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of and identify therapeutic targets.
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http://dx.doi.org/10.1073/pnas.2010428117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7777036PMC
December 2020

ReVac: a reverse vaccinology computational pipeline for prioritization of prokaryotic protein vaccine candidates.

BMC Genomics 2019 Dec 16;20(1):981. Epub 2019 Dec 16.

Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Background: Reverse vaccinology accelerates the discovery of potential vaccine candidates (PVCs) prior to experimental validation. Current programs typically use one bacterial proteome to identify PVCs through a filtering architecture using feature prediction programs or a machine learning approach. Filtering approaches may eliminate potential antigens based on limitations in the accuracy of prediction tools used. Machine learning approaches are heavily dependent on the selection of training datasets with experimentally validated antigens (positive control) and non-protective-antigens (negative control). The use of one or few bacterial proteomes does not assess PVC conservation among strains, an important feature of vaccine antigens.

Results: We present ReVac, which implements both a panoply of feature prediction programs without filtering out proteins, and scoring of candidates based on predictions made on curated positive and negative control PVCs datasets. ReVac surveys several genomes assessing protein conservation, as well as DNA and protein repeats, which may result in variable expression of PVCs. ReVac's orthologous clustering of conserved genes, identifies core and dispensable genome components. This is useful for determining the degree of conservation of PVCs among the population of isolates for a given pathogen. Potential vaccine candidates are then prioritized based on conservation and overall feature-based scoring. We present the application of ReVac, applied to 69 Moraxella catarrhalis and 270 non-typeable Haemophilus influenzae genomes, prioritizing 64 and 29 proteins as PVCs, respectively.

Conclusion: ReVac's use of a scoring scheme ranks PVCs for subsequent experimental testing. It employs a redundancy-based approach in its predictions of features using several prediction tools. The protein's features are collated, and each protein is ranked based on the scoring scheme. Multi-genome analyses performed in ReVac allow for a comprehensive overview of PVCs from a pan-genome perspective, as an essential pre-requisite for any bacterial subunit vaccine design. ReVac prioritized PVCs of two human respiratory pathogens, identifying both novel and previously validated PVCs.
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http://dx.doi.org/10.1186/s12864-019-6195-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916091PMC
December 2019

Differential functional patterns of memory CD4 and CD8 T-cells from volunteers immunized with Ty21a typhoid vaccine observed using a recombinant Escherichia coli system expressing S. Typhi proteins.

Vaccine 2020 01 16;38(2):258-270. Epub 2019 Oct 16.

Center for Vaccine Development and Global Health (CVD), Department of Pediatrics, University of Maryland School of Medicine, 685 West Baltimore Street, HSF1, Baltimore, MD 21201, USA.

It is widely accepted that CD4 and CD8 T-cells play a significant role in protection against Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever. However, the antigen specificity of these T-cells remains largely unknown. Previously, we demonstrated the feasibility of using a recombinant Escherichia coli (E. coli) expression system to uncover the antigen specificity of CD4 and CD8 T cells. Here, we expanded these studies to include the evaluation of 12 additional S. Typhi proteins: 4 outer membrane proteins (OmpH, OmpL, OmpR, OmpX), 3 Vi-polysaccharide biosynthesis proteins (TviA, TviB, TviE), 3 cold shock proteins (CspA, CspB, CspC), and 2 conserved hypothetical proteins (Chp 1 and Chp2), all selected based on the bioinformatic analyses of the content of putative T-cell epitopes. CD4 and CD8 T cells from 15 adult volunteers, obtained before and 42 days after immunization with oral live attenuated Ty21a vaccine, were assessed for their functionality (i.e., production of cytokines and cytotoxic expression markers in response to stimulation with selected antigens) as measured by flow cytometry. Although volunteers differed on their T-cell antigen specificity, we observed T-cell immune responses against all S. Typhi proteins evaluated. These responses included 9 proteins, OmpH, OmpR, TviA, TviE, CspA, CspB, CspC, Chp 1 and Chp 2, which have not been previously reported to elicit T-cell responses. Interestingly, we also observed that, regardless of the protein, the functional patterns of the memory T-cells were different between CD4 and CD8 T cells. In sum, these studies demonstrated the feasibility of using bioinformatic analysis and the E. coli expressing system described here to uncover novel immunogenic T-cell proteins that could serve as potential targets for the production of protein-based vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2019.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994170PMC
January 2020

Erratum for Kilian and Tettelin, "Identification of Virulence-Associated Properties by Comparative Genome Analysis of Streptococcus pneumoniae, , S. mitis, Three S. oralis Subspecies, and ".

mBio 2019 Oct 15;10(5). Epub 2019 Oct 15.

Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.

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http://dx.doi.org/10.1128/mBio.02520-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794489PMC
October 2019

Identification of Virulence-Associated Properties by Comparative Genome Analysis of Streptococcus pneumoniae, S. pseudopneumoniae, S. mitis, Three S. oralis Subspecies, and .

mBio 2019 09 3;10(5). Epub 2019 Sep 3.

Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.

From a common ancestor, and evolved in parallel into one of the most important pathogens and a mutualistic colonizer of humans, respectively. This evolutionary scenario provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. We performed detailed comparisons of 60 genomes of , , , the three subspecies , , and , and Nonfunctional remnants of ancestral genes in both and in support the evolutionary model and the concept that evolutionary changes on both sides were required to reach their present relationship to the host. Confirmed by screening of >7,500 genomes, we identified 224 genes associated with virulence. The striking difference to commensal streptococci was the diversity of regulatory mechanisms, including regulation of capsule production, a significantly larger arsenal of enzymes involved in carbohydrate hydrolysis, and proteins known to interfere with innate immune factors. The exclusive presence of the virulence factors in enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. In addition to loss of these virulence-associated genes, adaptation of to a mutualistic relationship with the host apparently required preservation or acquisition of 25 genes lost or absent from Successful adaptation of and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms. is one of the most important human pathogens but is closely related to , with which humans live in harmony. The fact that the two species evolved from a common ancestor provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. By detailed comparisons of genomes of the two species and other related streptococci, we identified 224 genes associated with virulence and 25 genes unique to the mutualistic species. The exclusive presence of the virulence factors in enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. Successful adaptation of and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms.
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http://dx.doi.org/10.1128/mBio.01985-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722419PMC
September 2019

Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination.

Elife 2018 12 28;7. Epub 2018 Dec 28.

MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

Pneumococcal whole cell vaccines (WCVs) could cost-effectively protect against a greater strain diversity than current capsule-based vaccines. Immunoglobulin G (IgG) responses to a WCV were characterised by applying longitudinally-sampled sera, available from 35 adult placebo-controlled phase I trial participants, to a panproteome microarray. Despite individuals maintaining distinctive antibody 'fingerprints', responses were consistent across vaccinated cohorts. Seventy-two functionally distinct proteins were associated with WCV-induced increases in IgG binding. These shared characteristics with naturally immunogenic proteins, being enriched for transporters and cell wall metabolism enzymes, likely unusually exposed on the unencapsulated WCV's surface. Vaccine-induced responses were specific to variants of the diverse PclA, PspC and ZmpB proteins, whereas PspA- and ZmpA-induced antibodies recognised a broader set of alleles. Temporal variation in IgG levels suggested a mixture of anamnestic and novel responses. These reproducible increases in IgG binding to a limited, but functionally diverse, set of conserved proteins indicate WCV could provide species-wide immunity.

Clinical trial registration: The trial was registered with ClinicalTrials.gov with Identifier NCT01537185; the results are available from https://clinicaltrials.gov/ct2/show/results/NCT01537185.
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http://dx.doi.org/10.7554/eLife.37015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344088PMC
December 2018

Using Core Genome Alignments To Assign Bacterial Species.

mSystems 2018 Nov-Dec;3(6). Epub 2018 Dec 4.

Institute for Genome Sciences, University of Maryland Baltimore, Baltimore, Maryland, USA.

With the exponential increase in the number of bacterial taxa with genome sequence data, a new standardized method to assign species designations is needed that is consistent with classically obtained taxonomic analyses. This is particularly acute for unculturable, obligate intracellular bacteria with which classically defined methods, like DNA-DNA hybridization, cannot be used, such as those in the . In this study, we generated nucleotide-based core genome alignments for a wide range of genera with classically defined species, as well as those within the . We created a workflow that uses the length, sequence identity, and phylogenetic relationships inferred from core genome alignments to assign genus and species designations that recapitulate classically obtained results. Using this method, most classically defined bacterial genera have a core genome alignment that is ≥10% of the average input genome length. Both and fail to meet this criterion, indicating that the taxonomy of these genera should be reexamined. Consistently, genomes from organisms with the same species epithet have ≥96.8% identity of their core genome alignments. Additionally, these core genome alignments can be used to generate phylogenomic trees to identify monophyletic clades that define species and neighbor-network trees to assess recombination across different taxa. By these criteria, organisms are delineated into species different from the currently used supergroup designations, while organisms are delineated into 9 distinct species, compared to the current 27 species. By using core genome alignments to assign taxonomic designations, we aim to provide a high-resolution, robust method to guide bacterial nomenclature that is aligned with classically obtained results. With the increasing availability of genome sequences, we sought to develop and apply a robust, portable, and high-resolution method for the assignment of genera and species designations that can recapitulate classically defined taxonomic designations. Using cutoffs derived from the lengths and sequence identities of core genome alignments along with phylogenetic analyses, we sought to evaluate or reevaluate genus- and species-level designations for diverse taxa, with an emphasis on the order , where species designations have been applied inconsistently. Our results indicate that the genus has an overabundance of species designations, that the current and genus designations are both too broad and need to be divided, and that there are clear demarcations of species that do not align precisely with the existing supergroup designations.
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http://dx.doi.org/10.1128/mSystems.00236-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280431PMC
December 2018

Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

Proc Natl Acad Sci U S A 2018 12 3;115(51):E12005-E12014. Epub 2018 Dec 3.

Institute of Human Virology, School of Medicine, University of Maryland, Baltimore, MD 21201;

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53 SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.
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http://dx.doi.org/10.1073/pnas.1815660115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304983PMC
December 2018

Persistence of Moraxella catarrhalis in Chronic Obstructive Pulmonary Disease and Regulation of the Hag/MID Adhesin.

J Infect Dis 2019 04;219(9):1448-1455

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore.

Background: Persistence of bacterial pathogens in the airways has profound consequences on the course and pathogenesis of chronic obstructive pulmonary disease (COPD). Patients with COPD continuously acquire and clear strains of Moraxella catarrhalis, a major pathogen in COPD. Some strains are cleared quickly and some persist for months to years. The mechanism of the variability in duration of persistence is unknown.

Methods: Guided by genome sequences of selected strains, we studied the expression of Hag/MID, hag/mid gene sequences, adherence to human cells, and autoaggregation in longitudinally collected strains of M. catarrhalis from adults with COPD.

Results: Twenty-eight of 30 cleared strains of M. catarrhalis expressed Hag/MID whereas 17 of 30 persistent strains expressed Hag/MID upon acquisition by patients. All persistent strains ceased expression of Hag/MID during persistence. Expression of Hag/MID in human airways was regulated by slipped-strand mispairing. Virulence-associated phenotypes (adherence to human respiratory epithelial cells and autoaggregation) paralleled Hag/MID expression in airway isolates.

Conclusions: Most strains of M. catarrhalis express Hag/MID upon acquisition by adults with COPD and all persistent strains shut off expression during persistence. These observations suggest that Hag/MID is important for initial colonization by M. catarrhalis and that cessation of expression facilitates persistence in COPD airways.
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http://dx.doi.org/10.1093/infdis/jiy680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467191PMC
April 2019

Changes in IgA Protease Expression Are Conferred by Changes in Genomes during Persistent Infection by Nontypeable Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

Infect Immun 2018 08 23;86(8). Epub 2018 Jul 23.

Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York, USA

Nontypeable (NTHi) is an exclusively human pathobiont that plays a critical role in the course and pathogenesis of chronic obstructive pulmonary disease (COPD). NTHi causes acute exacerbations of COPD and also causes persistent infection of the lower airways. NTHi expresses four IgA protease variants (A1, A2, B1, and B2) that play different roles in virulence. Expression of IgA proteases varies among NTHi strains, but little is known about the frequency and mechanisms by which NTHi modulates IgA protease expression during infection in COPD. To assess expression of IgA protease during natural infection in COPD, we studied IgA protease expression by 101 persistent strains (median duration of persistence, 161 days; range, 2 to 1,422 days) collected longitudinally from patients enrolled in a 20-year study of COPD upon initial acquisition and immediately before clearance from the host. Upon acquisition, 89 (88%) expressed IgA protease. A total of 16 of 101 (16%) strains of NTHi altered expression of IgA protease during persistence. Indels and slipped-strand mispairing of mononucleotide repeats conferred changes in expression of , , and Strains with underwent frequent changes in expression of IgA protease B2 during persistence, mediated by slipped-strand mispairing of a 7-nucleotide repeat, TCAAAAT, within the open reading frame of We conclude that changes in gene sequences result in changes in expression of IgA proteases by NTHi during persistent infection in the respiratory tract of patients with COPD.
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http://dx.doi.org/10.1128/IAI.00313-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056860PMC
August 2018

genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

Proc Natl Acad Sci U S A 2018 04 19;115(14):E3256-E3265. Epub 2018 Mar 19.

Department of Microbiology and Immunology, University at Buffalo, The State University of New York, Buffalo, NY 14203;

Nontypeable (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.
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http://dx.doi.org/10.1073/pnas.1719654115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889651PMC
April 2018

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

Infect Immun 2018 01 19;86(1). Epub 2017 Dec 19.

Department of Pediatrics, New York University, New York, New York, USA

(group B [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 () operon. Among the non- genes identified as conditionally essential was , 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 knockout strains demonstrated decreased expression of β-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Furthermore, chemical activation of the stringent response with serine hydroxamate increased β-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.
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http://dx.doi.org/10.1128/IAI.00612-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736797PMC
January 2018

Whole-Genome Sequences of Bacteremia Isolates of .

Genome Announc 2017 Sep 28;5(39). Epub 2017 Sep 28.

Department of Pediatrics, Columbia University, New York, New York, USA

causes respiratory and invasive diseases in humans, but its pathogenesis remains poorly understood. We report here the genome sequences of seven bacteremia isolates of , including the type strain. Comparative analysis of these sequences may aid studies of biology and assist in the development of species-specific diagnostic strategies.
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http://dx.doi.org/10.1128/genomeA.01023-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624759PMC
September 2017

Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells.

PLoS Negl Trop Dis 2017 Sep 5;11(9):e0005912. Epub 2017 Sep 5.

Center for Vaccine Development (CVD), Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States of America.

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in S. Typhi protection, it is crucial to identify S. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant Escherichia coli (E. coli) expressing one of four S. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific S. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant E. coli also possess the ability to respond to purified proteins. We observed that CD4+ cell responses, but not CD8+ cell responses, to recombinant E. coli were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an E. coli expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4+ and CD8+ cell responses.
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http://dx.doi.org/10.1371/journal.pntd.0005912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600385PMC
September 2017

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

PLoS Pathog 2017 Aug 25;13(8):e1006582. Epub 2017 Aug 25.

Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1006582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589263PMC
August 2017

Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.

PLoS Pathog 2017 Aug 23;13(8):e1006584. Epub 2017 Aug 23.

Department of Cell Biology & Molecular Genetics and Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, United States of America.

The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T0) mutant pools were calculated for each gene. We identified a total of 273 subcutaneous fitness (scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.
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http://dx.doi.org/10.1371/journal.ppat.1006584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584981PMC
August 2017

Whole-Exome Sequencing Identifies the 6q12-q16 Linkage Region and a Candidate Gene, TTK, for Pulmonary Nontuberculous Mycobacterial Disease.

Am J Respir Crit Care Med 2017 12;196(12):1599-1604

1 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.

Rationale: Pulmonary nontuberculous mycobacterial disease (PNTM) often affects white postmenopausal women, with a tall and lean body habitus and higher rates of scoliosis, pectus excavatum, mitral valve prolapse, and mutations in the CFTR gene. These clinical features and the familial clustering of the disease suggest an underlying genetic mechanism.

Objectives: To map the genes associated with PNTM, whole-exome sequencing was conducted in 12 PNTM families and 57 sporadic cases recruited at the National Institutes of Health Clinical Center during 2001-2013.

Methods: We performed a variant-level and a gene-level parametric linkage analysis on nine PNTM families (16 affected and 20 unaffected) as well as a gene-level association analysis on nine PNTM families and 55 sporadic cases.

Measurements And Main Results: The genome-wide variant-level linkage analysis using 4,328 independent common variants identified a 20-cM region on chromosome 6q12-6q16 (heterogeneity logarithm of odds score = 3.9), under a recessive disease model with 100% penetrance and a risk allele frequency of 5%. All genes on chromosome 6 were then tested in the gene-level linkage analysis, using the collapsed haplotype pattern method. The TTK protein kinase gene (TTK) on chromosome 6q14.1 was the most significant (heterogeneity logarithm of odds score = 3.38). In addition, the genes MAP2K4, RCOR3, KRT83, IFNLR1, and SLC29A1 were associated with PNTM in our gene-level association analysis.

Conclusions: The TTK gene encodes a protein kinase that is essential for mitotic checkpoints and the DNA damage response. TTK and other genetic loci identified in our study may contribute to the increased susceptibility to NTM infection and its progression to pulmonary disease.
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http://dx.doi.org/10.1164/rccm.201612-2479OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754439PMC
December 2017

CloVR-Comparative: automated, cloud-enabled comparative microbial genome sequence analysis pipeline.

BMC Genomics 2017 04 27;18(1):332. Epub 2017 Apr 27.

Institute for Genome Sciences, Baltimore, MD, USA.

Background: The benefit of increasing genomic sequence data to the scientific community depends on easy-to-use, scalable bioinformatics support. CloVR-Comparative combines commonly used bioinformatics tools into an intuitive, automated, and cloud-enabled analysis pipeline for comparative microbial genomics.

Results: CloVR-Comparative runs on annotated complete or draft genome sequences that are uploaded by the user or selected via a taxonomic tree-based user interface and downloaded from NCBI. CloVR-Comparative runs reference-free multiple whole-genome alignments to determine unique, shared and core coding sequences (CDSs) and single nucleotide polymorphisms (SNPs). Output includes short summary reports and detailed text-based results files, graphical visualizations (phylogenetic trees, circular figures), and a database file linked to the Sybil comparative genome browser. Data up- and download, pipeline configuration and monitoring, and access to Sybil are managed through CloVR-Comparative web interface. CloVR-Comparative and Sybil are distributed as part of the CloVR virtual appliance, which runs on local computers or the Amazon EC2 cloud. Representative datasets (e.g. 40 draft and complete Escherichia coli genomes) are processed in <36 h on a local desktop or at a cost of <$20 on EC2.

Conclusions: CloVR-Comparative allows anybody with Internet access to run comparative genomics projects, while eliminating the need for on-site computational resources and expertise.
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http://dx.doi.org/10.1186/s12864-017-3717-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408420PMC
April 2017

Complete Genome Sequences of Strains Isolated from Rhesus Macaques.

Genome Announc 2017 Apr 20;5(16). Epub 2017 Apr 20.

Department of Pathology, and Department of Epidemiology and Public Health, Program of Comparative Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

is a nontuberculous mycobacterium. It causes opportunistic infections with pulmonary and extrapulmonary manifestations. We report here the complete genome sequences of two strains isolated from rhesus macaques. We performed genome comparisons with human and environmental isolates of to assess the genomic diversity of this species.
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http://dx.doi.org/10.1128/genomeA.00187-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399255PMC
April 2017

Transcriptional organization of pneumococcal psrP-secY2A2 and impact of GtfA and GtfB deletion on PsrP-associated virulence properties.

Microbes Infect 2017 06 10;19(6):323-333. Epub 2017 Apr 10.

Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA; Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address:

Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence.
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http://dx.doi.org/10.1016/j.micinf.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581956PMC
June 2017

Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae.

mBio 2016 11 15;7(6). Epub 2016 Nov 15.

Department of Biomedicine, Aarhus University, Aarhus, Denmark

Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus) and S. infantis Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps) loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen.

Importance: Expression of a capsular polysaccharide is among the principal virulence factors of Streptococcus pneumoniae and is the basis for successful vaccines against infections caused by this important pathogen. Contrasting with previous assumptions, this study showed that expression of capsular polysaccharides by the same genetic mechanisms is a general property of closely related species of streptococci that form a significant part of our commensal microbiota. The demonstrated antigenic identity of many capsular polysaccharides expressed by commensal streptococci and S. pneumoniae raises important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and the pathogen.
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http://dx.doi.org/10.1128/mBio.01844-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111408PMC
November 2016

In-silico prediction and deep-DNA sequencing validation indicate phase variation in 115 Neisseria meningitidis genes.

BMC Genomics 2016 10 28;17(1):843. Epub 2016 Oct 28.

GSK Vaccines, 53100, Siena, Italy.

Background: The Neisseria meningitidis (Nm) chromosome shows a high abundance of simple sequence DNA repeats (SSRs) that undergo stochastic, reversible mutations at high frequency. This mechanism is reflected in an extensive phenotypic diversity that facilitates Nm adaptation to dynamic environmental changes. To date, phase-variable phenotypes mediated by SSRs variation have been experimentally confirmed for 26 Nm genes.

Results: Here we present a population-scale comparative genomic analysis that identified 277 genes and classified them into 52 strong, 60 moderate and 165 weak candidates for phase variation. Deep-coverage DNA sequencing of single colonies grown overnight under non-selective conditions confirmed the presence of high-frequency, stochastic variation in 115 of them, providing circumstantial evidence for their phase variability. We confirmed previous observations of a predominance of variable SSRs within genes for components located on the cell surface or DNA metabolism. However, in addition we identified an unexpectedly broad spectrum of other metabolic functions, and most of the variable SSRs were predicted to induce phenotypic changes by modulating gene expression at a transcriptional level or by producing different protein isoforms rather than mediating on/off translational switching through frameshifts. Investigation of the evolutionary history of SSR contingency loci revealed that these loci were inherited from a Nm ancestor, evolved independently within Nm, or were acquired by Nm through lateral DNA exchange.

Conclusions: Overall, our results have identified a broader and qualitatively different phenotypic diversification of SSRs-mediated stochastic variation than previously documented, including its impact on central Nm metabolism.
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http://dx.doi.org/10.1186/s12864-016-3185-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084427PMC
October 2016

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

Infect Immun 2016 10 19;84(10):2922-32. Epub 2016 Sep 19.

Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA Department of Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama, USA

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 β-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.
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http://dx.doi.org/10.1128/IAI.00277-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038079PMC
October 2016

The essential genome of Streptococcus agalactiae.

BMC Genomics 2016 05 26;17:406. Epub 2016 May 26.

Department of Pediatrics, Division of Pediatric Infectious Diseases, New York University School of Medicine, 550 First Avenue (MSB 223), New York, NY, 10016, USA.

Background: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis.

Methods: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries-with at least 135,000 mutants per library-at serial 24 h time points after outgrowth in rich media.

Results: After statistical analysis of transposon insertion density and distribution, we identified 13.5 % of genes as essential and 1.2 % as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93 % concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS.

Conclusions: We believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.
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http://dx.doi.org/10.1186/s12864-016-2741-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881062PMC
May 2016

Genome stability of Propionibacterium acnes: a comprehensive study of indels and homopolymeric tracts.

Sci Rep 2016 Feb 9;6:20662. Epub 2016 Feb 9.

Department of Biomedicine, Aarhus University, Denmark.

We present a species-wide comparative analysis of 90 genomes of Propionibacterium acnes that represent the known diversity of the species. Our results are augmented by six high-quality genomes and a manual investigation of all gene-sized indels found in the strains. Overall, the order of genes is conserved throughout the species. A public sybil database for easy comparative analysis of the 90 genomes was established. The analysis of indels revealed a total of 66 loci of non-core genes that correlate with phylogenetic clades. No gene was strain-specific in agreement with our conclusion that the P. acnes pan-genome is closed. An exhaustive search for homopolymeric tracts (HPTs) identified a total of 54 variable-length HPTs almost exclusively of guanine/cytosines located between genes or affecting the reading frame of genes. The repeat variation was consistent with phylogenetic clades suggesting slow accumulation over time rather than active modification. By transcriptome analysis we demonstrate how an HPT variation can affect the gene expression levels. Selected cases of both indels and HPTs are described. The catalogued data and the public P. acnes Sybil database provide a solid foundation for generating hypotheses and facilitate comparative genetic analyses in future P. acnes research.
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http://dx.doi.org/10.1038/srep20662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746626PMC
February 2016