A correlative study of the genomic underpinning of virulence traits and drug tolerance of Candida auris

Authors: Bo Yang, Benjamin Vaisvil, Daniel Schmitt, Joseph Collins, Eric Young, Vinayak Kapatral, Reeta Rao

Candida auris is an opportunistic fungal pathogen with high mortality rates which presents a clear threat to public health. The risk of C. auris infection is high because it can colonize the body, resist antifungal treatment, and evade the immune system. The genetic mechanisms for these traits are not well known. Identifying them could lead to new targets for new treatments. To this end, we present an analysis of the genetics and gene expression patterns of C. auris carbon metabolism, drug resistance, and macrophage interaction. We chose to study two C. auris isolates simultaneously, one drug sensitive (B11220 from Clade II) and one drug resistant (B11221 from Clade III). Comparing the genomes, we confirm the previously reported finding that B11220 was missing a 12.8 kb region on chromosome VI. This region contains a gene cluster encoding proteins related to alternative sugar utilization. We show that B11221, which has the gene cluster, readily assimilates and utilizes D-galactose and L-rhamnose as compared to B11220, which harbors the deletion. B11221 exhibits increased adherence and drug resistance compared to B11220 when grown in these sugars. Transcriptomic analysis of both isolates grown on glucose or galactose showed that the gene cluster was upregulated when grown on D-galactose. These findings reinforce growing evidence of a link between metabolism and drug tolerance. B11221 resists phagocytosis by macrophages and exhibits decreased β-1,3-glucan exposure, a key determinant that allows Candida to evade the host immune system, as compared to B11220. In a transcriptomic analysis of both isolates co-cultured with macrophages, we find upregulation of genes associated with transport and transcription factors in B11221. Our studies show a positive correlation between membrane composition and immune evasion, alternate sugar utilization, and drug tolerance in C. auris.

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

Binod Rayamajhee, Mark Willcox, Savitri Sharma, Ronnie Mooney, Constantinos Petsoglou, Paul R Badenoch, Samendra Sherchan, Fiona L Henriquez, Nicole Carnt

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II

Tom G. Schwan, Sandra J. Raffel, Stacy M. Ricklefs, Daniel P. Bruno, and Craig Martens

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined.

Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined.

Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86–95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3′ end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined.

Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

De novo assembly of Roseomonas mucosa isolated from patients with atopic dermatitis

Kent Barbian, Daniel Bruno, Lydia Sykora, Stacy Ricklefs, Prem Prashant Chaudhary, Paul A. Beare, Ian A. Myles, Craig M. Martens

ABSTRACT

Roseomonas mucosa is associated with the normal skin microflora. Here, we present de novo sequence assemblies from R. mucosa isolates obtained from the skin lesions of three atopic dermatitis patients.

ANNOUNCEMENT

Roseomonas mucosa is a Gram-negative coccobacillus found in aquatic environments and from various clinical samples (1 – 5). R. mucosa is commonly isolated from the microbiota of human skin (4, 6). Atopic dermatitis (AD), an inflammatory skin disease, causes susceptibility to Staphylococcus aureus infection, immune dysregulation function, and an impaired skin barrier. Improved AD clinical outcome was observed when R. mucosa was used to topically treat patients with AD (2, 4, 7). This treatment had an improved outcome in mouse models of AD. Interestingly, this mouse model treated with R. mucosa from AD patients, had no impact or a worse clinical outcome (2). We present the sequences from three AD-sourced R. mucosa isolates (2).

R. mucosa from lesions of three AD patients was isolated using a FloqSwab moistened in phosphate-buffered saline. Swabs were placed in Reasoner’s 2A (R2A) broth containing amphotericin B and vancomycin and incubated for 48–72 h at 32°C. Cultures were spread on R2A agar and incubated as above to form single colonies (4). Bacteria, stored at −80°C, were spread on R2A agar and a single colony used to inoculate 100 mls of R2A broth, and grown at 32°C for 20–25 h (8). Bacteria were washed, resuspended, and heated to 65°C, followed by separate incubations with proteinase k, lysozyme, and SDS. Each sample was incubated with RNaseI for 30 min at 37°C, then combined with a 3/4 vol of saturated phenol solution, mixed for 10 min and centrifugated at 12,800 × g for 10 min. The aqueous phase was removed and treated with phenol:chloroform:isoamyl alcohol (25:24:1) until the white precipitate interface was absent. A chloroform-only extraction was performed, and aqueous solution was collected. DNA was precipitated with 3 M sodium acetate, washed 2× with 70% EtOH, and resuspended in Qiagen’s EB buffer.

PacBio and Illumina sequencing libraries were generated from the same DNA using the 10 kb Template Preparation and Sequencing kit with Covaris g-tubes (sheared to >8,700 bp) and Illumina TruSeq Nano DNA kit using a Covaris M220 (sheared to ~550 bp), respectively (PacBio protocol 100-092-800-06 and TruSeq protocol 15041110 rev. D). Libraries were sequenced with PacBio Sequel and Illumina MiSeq (2 × 300 bp read length) platforms. Illumina reads were adapter trimmed using cutadapt (v.1.12) and trimmed and filtered for quality using FASTX-Toolkit (v.0.0.14). Bioinformatic analysis and construction of the genomes were performed using the following programs: CANU (v.1.0), Bowtie2 (v.2.2.4), pilon (v.1.16), and MIRA (v.4.0). All software was used with default parameters. The PacBio data were used to construct DNA scaffolds which were optimized using Illumina data. Igenbio Inc. annotated the genomes, while public annotation was compiled by the NCBI Prokaryotic Genome Annotation Pipeline (9).

The R. mucosa genomes consist of a single large circular chromosome (4.01–4.28 Mb) and five to six autonomously replicating plasmid sequences (8.9–506.3 kb) (Table 1). Genome properties and annotation statistics are listed in Table 1. These genomes may help determine the genes that are important for successful treatment of AD.

ACKNOWLEDGMENTS

We would like to thank Igenbio Inc. their sequencing services and data analysis.

This work was supported by funding from the Intramural Research Program of the NIH, NIAID 67 (Z01-147170). The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

The arginine deaminase system plays distinct roles in Borrelia burgdorferi and Borrelia hermsii

Crystal L. Richards, Sandra J. Raffel, Sebastien Bontemps-Gallo,Daniel P. Dulebohn, Tessa C. Herbert, Frank C. Gherardini

Borrelia species are amino acid auxotrophs that utilize di- and tri- peptides obtained through their oligopeptide transport system to supply amino acids for replicative growth during their enzootic cycles. However, Borrelia species from both the Lyme disease (LD) and relapsing fever (RF) groups harbor an amino acid transport and catabolism system, the Arginine Dei- minase System (ADI), that could potentially augment intracellular L-arginine required for growth. RF spirochetes contain a “complete”, four gene ADI (arcA, B, D, and C) while LD spirochetes harbor arcA, B, and sometimes D but lack arcC (encoding carbamate kinase). In this study, we evaluated the role of the ADI system in bacterial survival and virulence and discovered important differences in RF and LD ADIs. Both in vitro and in a murine model of infection, B. hermsii cells significantly reduced extracellular L-arginine levels and that reduc- tion was dependent on arginine deiminase expression. Conversely, B. burgdorferi did not reduce the concentration of L-arginine during in vitro growth experiments nor during infec- tion of the mammalian host, suggesting a fundamental difference in the ability to directly uti- lize L-arginine compared to B. hermsii. Further experiments using a panel of mutants generated in both B. burgdorferi and B. hermsii, identified important differences in growth characteristics and ADI transcription and protein expression. We also found that the ADI system plays a key role in blood and spleen colonization in RF spirochetes. In this study we have identified divergent metabolic strategies in two closely related human pathogens, that ultimately impacts the host-pathogen interface during infection.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism

Sébastien Bontemps-Gallo,1 Charlotte Gaviard,2,3 Crystal L. Richards,1 Takfarinas Kentache,2,3 Sandra J. Raffel,1 Kevin A. Lawrence,1 Joseph C. Schindler,4 Joseph Lovelace,4 Daniel P. Dulebohn,1 Robert G. Cluss,4 Julie Hardouin,2,3 and Frank C. Gherardini1

1 Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States

2 CNRS UMR 6270 Polymères, Biopolymères, Surfaces Laboratory, Université de Rouen, Mont-Saint-Aignan, France

3 PISSARO Proteomic Facility, Institut de Recherche et d’Innovation Biomédicale, Mont-Saint-Aignan, France

4 Department of Chemistry and Biochemistry, Middlebury College, Middlebury, VT, United States

This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

Genome Sequence and Comparative Pathogenic Determinants of Multidrug Resistant Uropathogenic Escherichia coli O25b:H4, A Clinical Isolate from Saudi Arabia

Essam J. Alyamani, Anamil M. Khiyami, Rayan Y. Booq, Fayez S. Bahwerth, Benjamin Vaisvil, Daniel P. Schmitt and Vinayak Kapatral

Escherichia coli serotype O25b:H4 is involved in human urinary tract infections. In this study, we sequenced and analyzed E. coli O25b:H4 isolated from a patient suffering from recurring UTI infections in an intensive care unit at Hera General Hospital in Makkah, Saudi Arabia. We aimed to determine the virulence genes for pathogenesis and drug resistance of this isolate compared to other E. coli strains. We sequenced and analyzed the E. coli O25b:H4 Saudi strain clinical isolate using next generation sequencing. Using the ERGO genome analysis platform, we performed annotations and identified virulence and antibiotic resistance determinants of this clinical isolate. The E. coli O25b:H4 genome was assembled into four contigs representing a total chromosome size of 5.28 Mb, and three contigs were identified, including a 130.9 kb (virulence plasmid) contig bearing the bla-CTX gene and 32 kb and 29 kb contigs. In comparing this genome to other uropathogenic E. coli genomes, we identified unique drug resistance and pathogenicity factors. In this work, whole-genome sequencing and targeted comparative analysis of a clinical isolate of uropathogenic Escherichia coli O25b:H4 was performed. This strain encodes virulence genes linked with extraintestinal pathogenic E. coli (ExPEC) that are expressed constitutively in E. coli ST131. We identified the genes responsible for pathogenesis and drug resistance and performed comparative analyses of the virulence and antibiotic resistance determinants with those of other E. coli UPEC isolates. This is the first report of genome sequencing and analysis of a UPEC strain from Saudi Arabia.

Published Nov 5. 2016. DOI: 10.22207/JPAM.10.4.01

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection

Hannah K. Wilder, Sandra J. Raffel, Alan G. Barbour, Stephen F. Porcella, Daniel E. Sturdevant, Benjamin Vaisvil, Vinayak Kapatral, Daniel P. Schmitt, Tom G. Schwan, Job E. Lopez

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Published: February 4, 2016 DOI: 10.1371/journal.pone.0147707

The genome of Shigella dysenteriae strain Sd1617 comparison to representative strains in evaluating pathogenesis

Ajchara A. Vongsawan , Vinayak Kapatral , Benjamin Vaisvil , Henry Burd , Oralak Serichantalergs , Malabi M. Venkatesan , Carl J. Mason

We sequenced and analyzed Shigella dysenteriae strain Sd1617 serotype 1 that is widely used as model strain for vaccine design, trials and research. A combination of next-generation sequencing platforms and assembly yielded two contigs representing a chromosome size of 4.34 Mb and the large virulence plasmid of 177 kb. This genome sequence is compared with other Shigella genomes in order to understand gene complexity and pathogenic factors.

FEMS Microbiology Letters, 362, 2015, fnv011 doi: 10.1093/femsle/fnv011

Genomic Avenue to Avian Colisepticemia

Sagi Huja, Yaara Oren, Eva Trost, Elzbieta Brzuszkiewicz, Dvora Biran,a Jochen Blom, Alexander Goesmann, Gerhard Gottschalk, Jörg Hacker, Eliora Z. Ron, Ulrich Dobrindt

ABSTRACT Here we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenic Escherichia coli (APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78—O78:H19 ST88 isolate 789 (O78-9)—and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. coli type 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of the Yersinia high-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum. IMPORTANCE Avian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.

Complete Genome Sequence of Flavobacterium psychrophilum Strain CSF259-93, Used To Select Rainbow Trout for Increased Genetic Resistance against Bacterial Cold Water Disease.

Wiens GD, LaPatra SE, Welch TJ, Rexroad C 3rd, Call DR, Cain KD, LaFrentz BR, Vaisvil B, Schmitt DP, Kapatral V.

The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increased genetic resistance against bacterial cold water disease.

Genome Announc. 2014 Sep 18;2(5). pii: e00889-14. doi: 10.1128/genomeA.00889-14.

Draft Genome Sequence of a New Homofermentative, Lactic AcidProducing Enterococcus faecalis Isolate, CBRD01

Lew P. Christopher, Vinayak Kapatral, Benjamin Vaisvil, Ginger Emel,b and Linda C. DeVeauxc

We report here the draft genome sequence of the novel homofermentative Enterococcus faecalis isolate CBRD01, which is capable of high lactic acid productivity and yields, with minimal nutritional requirements. The genome is 2.8 Mbp, with 37% G+C, and contains genes for two lactate dehydrogenase (LDH) enzymes found in related organisms.

Genome Announc. 2014 Mar-Apr; 2(2): e00147-14.
Published online 2014 Mar 27. doi:  10.1128/genomeA.00147-14

Metabolic Network Analysis-Based Identification of Antimicrobial Drug Targets in Category A Bioterrorism Agents

Yong-Yeol Ahn, Deok-Sun Lee, Henry Burd, William Blank, Vinayak Kapatral 

The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents.

Published: January 15, 2014DOI: 10.1371/journal.pone.0085195

Comparative Genomics and Transcriptomics of Propionibacterium acnes

Elzbieta Brzuszkiewicz, January Weiner, Antje Wollherr, Andrea Thürmer,Jennifer Hüpeden, Hans B. Lomholt,Mogens Kilian, Gerhard Gottschalk, Rolf Daniel, Hans-Joachim Mollenkopf, Thomas F. Meyer, Holger Brüggemann

The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with maintenance of skin homeostasis. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits differ between phylotypes, as judged from PCR analysis of a collection of P. acnes strains. Comparative transcriptome analysis of strains KPA171202 (type I-2) and 266 during exponential growth revealed inter-strain differences in gene expression of transport systems and metabolic pathways. In addition, transcript levels of genes encoding possible virulence factors such as dermatan-sulphate adhesin, polyunsaturated fatty acid isomerase, iron acquisition protein HtaA and lipase GehA were upregulated in strain 266. We investigated differential gene expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by upregulation of genes involved in stress responses and amino acid metabolism. Our data highlight the genomic basis for strain diversity and identify, for the first time, the actively transcribed part of the genome, underlining the important role growth status plays in the inflammation-inducing activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by the phylotype-specific genome content but also by variable gene expression.

Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3.

Batzilla J, Höper D, Antonenka U, Heesemann J, Rakin A.

We report here the first finished and annotated genome sequence of a representative of the most epidemiologically successful Yersinia group, Y. enterocolitica subsp. palearctica strain Y11, serotype O:3, biotype 4. This strain is a certified type strain of the German DSMZ collection (DSM no. 13030; Yersinia enterocolitica subsp. palearctica) that was isolated from the stool of a human patient (H. Neubauer, S. Aleksic, A. Hensel, E. J. Finke, and H. Meyer. Int. J. Med. Microbiol. 290:61-64, 2000).

J Bacteriol. 2011 Apr;193(8):2067. doi: 10.1128/JB.01484-10. Epub 2011 Feb 4.

Blueprint for antimicrobial hit discovery targeting metabolic networks.

Shen Y, Liu J, Estiu G, Isin B, Ahn YY, Lee DS, Barabási AL, Kapatral V, Wiest O, Oltvai ZN.

Advances in genome analysis, network biology, and computational chemistry have the potential to revolutionize drug discovery by combining system-level identification of drug targets with the atomistic modeling of small molecules capable of modulating their activity. To demonstrate the effectiveness of such a discovery pipeline, we deduced common antibiotic targets in Escherichia coli and Staphylococcus aureus by identifying shared tissue-specific or uniformly essential metabolic reactions in their metabolic networks. We then predicted through virtual screening dozens of potential inhibitors for several enzymes of these reactions and showed experimentally that a subset of these inhibited both enzyme activities in vitro and bacterial cell viability. This blueprint is applicable for any sequenced organism with high-quality metabolic reconstruction and suggests a general strategy for strain-specific antiinfective therapy.

Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1082-7. doi: 10.1073/pnas.0909181107. Epub 2010 Jan 5.

Comparative Genome-Scale Metabolic Reconstruction and Flux Balance Analysis of Multiple Staphylococcus aureus Genomes Identify Novel Antimicrobial Drug Targets

Deok-Sun Lee, Henry Burd, Jiangxia Liu, Eivind Almaas, Olaf Wiest, Albert-László Barabási, Zoltán N. Oltvai, and  Vinayak Kapatral

Mortality due to multidrug-resistant Staphylococcus aureus infection is predicted to surpass that of human immunodeficiency virus/AIDS in the United States. Despite the various treatment options for S. aureusinfections, it remains a major hospital- and community-acquired opportunistic pathogen. With the emergence of multidrug-resistant S. aureus strains, there is an urgent need for the discovery of new antimicrobial drug targets in the organism. To this end, we reconstructed the metabolic networks of multidrug-resistant S. aureus strains using genome annotation, functional-pathway analysis, and comparative genomic approaches, followed by flux balance analysis-based in silico single and double gene deletion experiments. We identified 70 single enzymes and 54 pairs of enzymes whose corresponding metabolic reactions are predicted to be unconditionally essential for growth. Of these, 44 single enzymes and 10 enzyme pairs proved to be common to all 13 S. aureus strains, including many that had not been previously identified as being essential for growth by gene deletion experiments in S. aureus. We thus conclude that metabolic reconstruction and in silico analyses of multiple strains of the same bacterial species provide a novel approach for potential antibiotic target identification.

J Bacteriol. 2009 Jun; 191(12): 4015–4024.
Published online 2009 Apr 17. doi:  10.1128/JB.01743-08

Host cell-free growth of the Q fever bacterium Coxiella burnetii.

Omsland A1, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA.

The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism's metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log(10)) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism's pathogenesis and genetics and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.

Proc Natl Acad Sci U S A. 2009 Mar 17;106(11):4430-4. doi: 10.1073/pnas.0812074106. Epub 2009 Feb 25.

Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella.

Beare PA1, Unsworth N, Andoh M, Voth DE, Omsland A, Gilk SD, Williams KP, Sobral BW, Kupko JJ 3rd, Porcella SF, Samuel JE, Heinzen RA.

Genetically distinct isolates of Coxiella burnetii, the cause of human Q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogenic potential, we deciphered the whole-genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structures of other bacterial pathogens that have recently emerged from nonpathogens with expanded niches. The observation that the attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests that this isolate's lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.

Infect Immun. 2009 Feb;77(2):642-56. doi: 10.1128/IAI.01141-08. Epub 2008 Dec 1.

Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

Wiens GD, Rockey DD, Wu Z, Chang J, Levy R, Crane S, Chen DS, Capri GR, Burnett JR, Sudheesh PS, Schipma MJ, Burd H, Bhattacharyya A, Rhodes LD, Kaul R, Strom MS.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

J Bacteriol. 2008 Nov;190(21):6970-82. 
doi: 10.1128/JB.00721-08. Epub 2008 Aug 22.