Adam A. Witney

A proteomic view of the Plasmodium falciparum life cycle.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context of Plasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuated P. falciparum sporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterized P. falciparum-derived protein antigens. The data suggest that immune responses to Plasmodium are dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens.

Evolutionary Genomics of Staphylococcus aureus Reveals Insights into the Origin and Molecular Basis of Ruminant Host Adaptation

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host–pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbpSov2), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Application of Comparative Phylogenomics To Study the Evolution of Yersinia enterocolitica and To Identify Genetic Differences Relating to Pathogenicity

Yersinia enterocolitica, an important cause of human gastroenteritis generally caused by the consumption of livestock, has traditionally been categorized into three groups with respect to pathogenicity, i.e., nonpathogenic (biotype 1A), low pathogenicity (biotypes 2 to 5), and highly pathogenic (biotype 1B). However, genetic differences that explain variation in pathogenesis and whether different biotypes are associated with specific nonhuman hosts are largely unknown. In this study, we applied comparative phylogenomics (whole-genome comparisons of microbes with DNA microarrays combined with Bayesian phylogenies) to investigate a diverse collection of 94 strains of Y. enterocolitica consisting of 35 human, 35 pig, 15 sheep, and 9 cattle isolates from nonpathogenic, low-pathogenicity, and highly pathogenic biotypes. Analysis confirmed three distinct statistically supported clusters composed of a nonpathogenic clade, a low-pathogenicity clade, and a highly pathogenic clade. Genetic differences revealed 125 predicted coding sequences (CDSs) present in all highly pathogenic strains but absent from the other clades. These included several previously uncharacterized CDSs that may encode novel virulence determinants including a hemolysin, a metalloprotease, and a type III secretion effector protein. Additionally, 27 CDSs were identified which were present in all 47 low-pathogenicity strains and Y. enterocolitica 8081 but absent from all nonpathogenic 1A isolates. Analysis of the core gene set for Y. enterocolitica revealed that 20.8% of the genes were shared by all of the strains, confirming this species as highly heterogeneous, adding to the case for the existence of three subspecies of Y. enterocolitica. Further analysis revealed that Y. enterocolitica does not cluster according to source (host).

A two‐component system that controls the expression of pneumococcal surface antigen A (PsaA) and regulates virulence and resistance to oxidative stress in Streptococcus pneumoniae

Recent genomic‐based studies have identified 13 two‐component signal transduction systems (TCS) in Streptococcus pneumoniae. Bacterial TCSs are important for regulating expression of bacterial genes, including those which are important to the virulence of pathogenic bacteria. We have used virulence assays together with microarray analysis to investigate the importance of pneumococcal TCS04 in the virulence and gene regulation of this pathogen. Deletion mutants of the response regulator of TCS04, rr04, were examined in three independent pneumococcal strains representing three different pneumococcal serotypes. Analysis of the virulence of the three strains enabled us to identify a serotype‐specific attenuation of virulence due to deletion of rr04. Microarray comparison of the transcriptional profiles of the wild‐type strains with the rr04 mutants allowed us to determine which transcriptional changes were occurring in the rr04 mutants. Virulence‐associated changes were demonstrated in the attenuated strain with significant downregulation of a previously determined virulence locus, psaB, psaC and psaA.

High Prevalence of Antibiotic-Resistant Mycoplasma genitalium in Nongonococcal Urethritis: The Need for Routine Testing and the Inadequacy of Current Treatment Options

Mycoplasma genitalium infections were as frequent a cause of nongonococcal urethritis as Chlamydia trachomatis, had high rates of macrolide-associated genotypic resistance, and were nonclonal, suggesting an established community infection. Detection of genotypic resistance to fluoroquinolones is cause for concern.

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

The Distribution of Mobile Genetic Elements (MGEs) in MRSA CC398 Is Associated with Both Host and Country

Methicillin-resistant Staphylococcus aureus clonal complex (CC) 398 has emerged from pigs to cause human infections in Europe and North America. We used a new 62-strain S. aureus microarray (SAM-62) to compare genomes of isolates from three geographical areas (Belgium, Denmark, and Netherlands) to understand how CC398 colonizes different mammalian hosts. The core genomes of 44 pig isolates and 32 isolates from humans did not vary. However, mobile genetic element (MGE) distribution was variable including SCCmec. φ3 bacteriophage and human specificity genes (chp, sak, scn) were found in invasive human but not pig isolates. SaPI5 and putative ruminant specificity gene variants (vwb and scn) were common but not pig specific. Virulence and resistance gene carriage was host associated but country specific. We conclude MGE exchange is frequent in CC398 and greatest among populations in close contact. This feature may help determine epidemiological associations among isolates of the same lineage.

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCα and PfGCβ) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCβ inEscherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCα was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases

ABSTRACT The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.