Stephanie L. Servetas

Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood.

BackgroundThe introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown.ResultsIn the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis.ConclusionThe analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.

Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

UNLABELLED Helicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ΔnikR ΔarsS, and Δfur ΔnikR ΔarsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation. IMPORTANCE Despite a paucity of regulatory proteins, adaptation is key to the survival of H. pylori within the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.

Helicobacter pylori bab Paralog Distribution and Association with cagA, vacA, and homA/B Genotypes in American and South Korean Clinical Isolates

Helicobacter pylori genetic variation is a crucial component of colonization and persistence within the inhospitable niche of the gastric mucosa. As such, numerous H. pylori genes have been shown to vary in terms of presence and genomic location within this pathogen. Among the variable factors, the Bab family of outer membrane proteins (OMPs) has been shown to differ within subsets of strains. To better understand genetic variation among the bab genes and to determine whether this variation differed among isolates obtained from different geographic locations, we characterized the distribution of the Bab family members in 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). Overall, we identified 23 different bab genotypes (19 in AH and 11 in KH), but only 5 occurred in greater than 5 isolates. Regardless of strain origin, a strain in which locus A and locus B were both occupied by a bab gene was the most common (85%); locus C was only occupied in those isolates that carried bab paralog at locus A and B. While the babA/babB/- genotype predominated in the KH (78.8%), no single genotype could account for greater than 40% in the AH collection. In addition to basic genotyping, we also identified associations between bab genotype and well known virulence factors cagA and vacA. Specifically, significant associations between babA at locus A and the cagA EPIYA-ABD motif (P<0.0001) and the vacA s1/i1/m1 allele (P<0.0001) were identified. Log-linear modeling further revealed a three-way association between bab carried at locus A, vacA, and number of OMPs from the HOM family (P<0.002). En masse this study provides a detailed characterization of the bab genotypes from two distinct populations. Our analysis suggests greater variability in the AH, perhaps due to adaptation to a more diverse host population. Furthermore, when considering the presence or absence of both the bab and homA/B paralogs at their given loci and the vacA genotype, an association was observed. Our results highlight the multifactorial nature of H. pylori mediated disease and the importance of considering how the specific combinations of H. pylori virulence genes and their multiple interactions with the host will collectively impact disease progression.

Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct “cross-talk” between HpArsRS and HpNikR at neutral pH has been demonstrated.

Analysis of a single Helicobacter pylori strain over a 10-year period in a primate model.

Helicobacter pylori from different individuals exhibits substantial genetic diversity. However, the kinetics of bacterial diversification after infection with a single strain is poorly understood. We investigated evolution of H. pylori following long-term infection in the primate stomach; Rhesus macaques were infected with H. pylori strain USU101 and then followed for 10 years. H. pylori was regularly cultured from biopsies, and single colony isolates were analyzed. At 1-year, DNA fingerprinting showed that all output isolates were identical to the input strain; however, at 5-years, different H. pylori fingerprints were observed. Microarray-based comparative genomic hybridization revealed that long term persistence of USU101 in the macaque stomach was associated with specific whole gene changes. Further detailed investigation showed that levels of the BabA protein were dramatically reduced within weeks of infection. The molecular mechanisms behind this reduction were shown to include phase variation and gene loss via intragenomic rearrangement, suggesting strong selective pressure against BabA expression in the macaque model. Notably, although there is apparently strong selective pressure against babA, babA is required for establishment of infection in this model as a strain in which babA was deleted was unable to colonize experimentally infected macaques.

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

Comparative analysis of the Hom family of outer membrane proteins in isolates from two geographically distinct regions: The United States and South Korea

Helicobacter pylori encodes numerous outer membrane proteins (OMPs), but only a few have been characterized in depth. Deletion, duplication, and allelic variation of many of the H. pylori OMPs have been reported, which suggests that these proteins may play key roles in host adaptation. Herein, we characterize the variation observed within the Hom family of OMPs in H. pylori obtained from two geographically distinct populations.

Helicobacter pylori outer membrane protein, HomC, shows geographic dependent polymorphism that is influenced by the Bab family

The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homCS, homCL, and homCM, in both populations. Similar to other polymorphic genes, the KH group showed less overall diversity, with 97.5% of strains harboring homCL. In contrast, a more heterogeneous profile was observed in strains derived from an American population; we found nearly equal distribution of homCS and homCL. Further analysis of the AH group identified associations between homC polymorphism and bab genotype; in AH strains, there was a significant association between homCL and carriage of babA at locus A. Since babA is an important virulence factor for the development of severe gastric disease, these data may suggest that homC polymorphism plays a role in H. pylori pathogenesis.

Certhrax Is an Antivirulence Factor for the Anthrax-Like Organism Bacillus cereus Strain G9241

ABSTRACT Bacillus cereus G9241 caused a life-threatening anthrax-like lung infection in a previously healthy human. This strain harbors two large virulence plasmids, pBCXO1 and pBC210, that are absent from typical B. cereus isolates. The pBCXO1 plasmid is nearly identical to pXO1 from Bacillus anthracis and carries genes (pagA1, lef, and cya) for anthrax toxin components (protective antigen [called PA1 in G9241], lethal factor [LF], and edema factor [EF], respectively). The plasmid also has an intact hyaluronic acid capsule locus. The pBC210 plasmid has a tetrasaccharide capsule locus, a gene for a PA1 homolog called PA2 (pagA2), and a gene (cer) for Certhrax, an ADP-ribosyltransferase toxin that inactivates vinculin. LF, EF, and Certhrax require PA for entry into cells. In this study, we asked what role PA1, PA2, LF, and Certhrax play in the pathogenicity of G9241. To answer this, we generated isogenic deletion mutations in the targeted toxin gene components and then assessed the strains for virulence in highly G9241-susceptible (A/J) and moderately G9241-sensitive (C57BL/6) mice. We found that full virulence of G9241 required PA1 and LF, while PA2 contributed minimally to pathogenesis of G9241 but could not functionally replace PA1 as a toxin-binding subunit in vivo. Surprisingly, we discovered that Certhrax attenuated the virulence of G9241; i.e., a Δcer Δlef mutant strain was more virulent than a Δlef mutant strain following subcutaneous inoculation of A/J mice. Moreover, the enzymatic activity of Certhrax contributed to this phenotype. We concluded that Certhrax acts as an antivirulence factor in the anthrax-like organism B. cereus G9241.

ArsRS-Dependent Regulation of homB Contributes to Helicobacter pylori Biofilm Formation

One elusive area in the Helicobacter pylori field is an understanding of why some infections result in gastric cancer, yet others persist asymptomatically for the life-span of the individual. Even before the genomic era, the high level of intraspecies diversity of H. pylori was well recognized and became an intriguing area of investigation with respect to disease progression. Of interest in this regard is the unique repertoire of over 60 outer membrane proteins (OMPs), several of which have been associated with disease outcome. Of these OMPs, the association between HomB and disease outcome varies based on the population being studied. While the molecular roles for some of the disease-associated OMPs have been evaluated, little is known about the role that HomB plays in the H. pylori lifecycle. Thus, herein we investigated homB expression, regulation, and contribution to biofilm formation. We found that in H. pylori strain G27, homB was expressed at a relatively low level until stationary phase. Furthermore, homB expression was suppressed at low pH in an ArsRS-dependent manner; mutation of arsRS resulted in increased homB transcript at all tested time-points. ArsRS regulation of homB appeared to be direct as purified ArsR was able to specifically bind to the homB promoter. This regulation, combined with our previous finding that ArsRS mutations lead to enhanced biofilm formation, led us to test the hypothesis that homB contributes to biofilm formation by H. pylori. Indeed, subsequent biofilm analysis using a crystal-violet quantification assay and scanning electron microscopy (SEM) revealed that loss of homB from hyper-biofilm forming strains resulted in reversion to a biofilm phenotype that mimicked wild-type. Furthermore, expression of homB in trans from a promoter that negated ArsRS regulation led to enhanced biofilm formation even in strains in which the chromosomal copy of homB had been deleted. Thus, homB is necessary for hyper-biofilm formation of ArsRS mutant strains and aberrant regulation of this gene is sufficient to induce a hyper-biofilm phenotype. In summary, these data suggest that the ArsRS-dependent regulation of OMPs such as HomB may be one mechanism by which ArsRS dictates biofilm development in a pH responsive manner.