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1.  NextGen sequencing reveals short double crossovers contribute disproportionately to genetic diversity in Toxoplasma gondii 
BMC Genomics  2014;15(1):1168.
Toxoplasma gondii is a widespread protozoan parasite of animals that causes zoonotic disease in humans. Three clonal variants predominate in North America and Europe, while South American strains are genetically diverse, and undergo more frequent recombination. All three northern clonal variants share a monomorphic version of chromosome Ia (ChrIa), which is also found in unrelated, but successful southern lineages. Although this pattern could reflect a selective advantage, it might also arise from non-Mendelian segregation during meiosis. To understand the inheritance of ChrIa, we performed a genetic cross between the northern clonal type 2 ME49 strain and a divergent southern type 10 strain called VAND, which harbors a divergent ChrIa.
NextGen sequencing of haploid F1 progeny was used to generate a genetic map revealing a low level of conventional recombination, with an unexpectedly high frequency of short, double crossovers. Notably, both the monomorphic and divergent versions of ChrIa were isolated with equal frequency. As well, ChrIa showed no evidence of being a sex chromosome, of harboring an inversion, or distorting patterns of segregation. Although VAND was unable to self fertilize in the cat, it underwent successful out-crossing with ME49 and hybrid survival was strongly associated with inheritance of ChrIII from ME49 and ChrIb from VAND.
Our findings suggest that the successful spread of the monomorphic ChrIa in the wild has not been driven by meiotic drive or related processes, but rather is due to a fitness advantage. As well, the high frequency of short double crossovers is expected to greatly increase genetic diversity among progeny from genetic crosses, thereby providing an unexpected and likely important source of diversity.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1168) contains supplementary material, which is available to authorized users.
PMCID: PMC4326188  PMID: 25532601
Gene conversion; Genetic mapping; Meiotic drive; Mendelian inheritance; Double crossover; Gene conversion
2.  Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development 
BMC Genomics  2014;15(1):350.
Considerable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression.
To profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo.
Merozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-350) contains supplementary material, which is available to authorized users.
PMCID: PMC4035076  PMID: 24885521
Toxoplasma gondii; Merozoite; Enteric stages; Definitive host; Life cycle; Parasite; Gene expression
3.  Compensatory dendritic cell development mediated by BATF-IRF interactions 
Nature  2012;490(7421):502-507.
The AP-1 transcription factor Batf3 is required for homeostatic development of CD8α+ classical dendritic cells that prime CD8 T-cell responses against intracellular pathogens. Here, we identify an alternative, Batf3-independent pathway for their development operating during infection with intracellular pathogens mediated by the cytokines IL-12 and IFN-γ. This alternative pathway results from molecular compensation for Batf3 provided by the related AP-1 factors Batf, which also functions in T and B cells, and Batf2 induced by cytokines in response to infection. Reciprocally, physiologic compensation between Batf and Batf3 also occurs in T cells for expression of IL-10 and CTLA-4. Compensation among BATF factors is based on the shared capacity of their leucine zipper domains to interact with non-AP-1 factors such as Irf4 and Irf8 to mediate cooperative gene activation. Conceivably, manipulating this alternative pathway of dendritic cell development could be of value in augmenting immune responses to vaccines.
PMCID: PMC3482832  PMID: 22992524
5.  MRSA Transmission on a Neonatal Intensive Care Unit: Epidemiological and Genome-Based Phylogenetic Analyses 
PLoS ONE  2013;8(1):e54898.
Methicillin-resistant Staphylococcus aureus (MRSA) may cause prolonged outbreaks of infections in neonatal intensive care units (NICUs). While the specific factors favouring MRSA spread on neonatal wards are not well understood, colonized infants, their relatives, or health-care workers may all be sources for MRSA transmission. Whole-genome sequencing may provide a new tool for elucidating transmission pathways of MRSA at a local scale.
Methods and Findings
We applied whole-genome sequencing to trace MRSA spread in a NICU and performed a case-control study to identify risk factors for MRSA transmission. MRSA genomes had accumulated sequence variation sufficiently fast to reflect epidemiological linkage among individual patients, between infants and their mothers, and between infants and staff members, such that the relevance of individual nurses’ nasal MRSA colonization for prolonged transmission could be evaluated. In addition to confirming previously reported risk factors, we identified an increased risk of transmission from infants with as yet unknown MRSA colonisation, in contrast to known MRSA-positive infants.
The integration of epidemiological (temporal, spatial) and genomic data enabled the phylogenetic testing of several hypotheses on specific MRSA transmission routes within a neonatal intensive-care unit. The pronounced risk of transmission emanating from undetected MRSA carriers suggested that increasing the frequency or speed of microbiological diagnostics could help to reduce transmission of MRSA.
PMCID: PMC3561456  PMID: 23382995
6.  The Polymorphic Pseudokinase ROP5 Controls Virulence in Toxoplasma gondii by Regulating the Active Kinase ROP18 
PLoS Pathogens  2012;8(11):e1002992.
Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.
Author Summary
The ability of microorganisms to cause disease in their hosts is often mediated by proteins that are secreted by the pathogen into the host cell as a means of disarming host signaling. Previous studies with the protozoan parasite Toxoplasma gondii have revealed that secretion of parasite protein kinases into the host cell mediates virulence in mouse, a natural host for transmission. Curiously, some of these virulence factors are active protein kinases, while other related pseudokinases lack enzymatic activity; hence, it was unclear how they functioned in promoting virulence. In the present work we demonstrate that ROP5, an inactive member of this protein kinase family, regulates the active protein kinase ROP18, which normally prevents clearance of the parasite in interferon-activated macrophages. Allosteric regulation of enzymes is a common theme in biology, but this is the first example of such a mechanism regulating a pathogen virulence factor. The potential advantage of such a layered process is that it might allow greater temporal or spatial control and perhaps protect the parasite from disabling strategies by the host.
PMCID: PMC3493473  PMID: 23144612
7.  Decreasing healthcare-associated infections (HAI) is an efficient method to decrease healthcare-associated Methicillin-resistant S.aureus (MRSA) infections Antimicrobial resistance data from the German national nosocomial surveillance system KISS 
By analysing the data of the intensive care unit (ICU) component of the German national nosocomial infection surveillance system (KISS) during the last ten years, we have observed a steady increase in the MRSA rates (proportions) from 2001 to 2005 and only a slight decrease from 2006 to 2010. The objective of this study was to investigate the development of the incidence density of nosocomial MRSA infections because this is the crucial outcome for patients.
Data from 103 ICUs with ongoing participation during the observation period were included. The pooled incidence density of nosocomial MRSA infections decreased significantly from 0.37 per 1000 patient days in 2001 to 0.15 per 1000 patient days in 2010 (RR = 0.40; CI95 0.29-0.55). This decrease was proportional to the significant decrease of all HCAI during the same time period (RR = 0.61; CI95 0.58-0.65).
The results underline the need to concentrate infection control activities on measures to control HCAI in general rather than focusing too much on specific MRSA prevention measures. MRSA rates (proportions) are not a very useful indicator of the situation.
PMCID: PMC3415117  PMID: 22958746
Surveillance; MRSA; epidemiology; Staphylococcus aureus
8.  Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression 
Molecular microbiology  2010;79(1):192-204.
Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility, and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signaling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short-term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.
PMCID: PMC3075969  PMID: 21166903
Egress; Invasion; Gene regulation; Microarray; Promoter; Synchronization
9.  Phosphorylation of immunity-related GTPases by a Toxoplasma gondii secreted kinase promotes macrophage survival and virulence 
Cell host & microbe  2010;8(6):484-495.
Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.
PMCID: PMC3013631  PMID: 21147463
10.  A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor 
PLoS Pathogens  2011;7(12):e1002392.
Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.
Author Summary
Apicomplexa are a group of parasites that cause important diseases, including malaria and several AIDS associated opportunistic infections. The parasites depend on an algal endosymbiont, the apicoplast, and this provides an Achilles' heel for drug development. We use Toxoplasma gondii as a model to characterize the biology and function of the apicoplast. In this study we apply a strategy to identify new apicoplast proteins and to prioritize them as potential targets through the analysis of genetic mutants. To aid this goal we develop a new parasite line and a protocol enabling the streamlined construction of conditional mutants. Using this new approach we discover numerous new apicoplast proteins, many of them have no assigned function yet. We demonstrate that function can be deduced using our genetic approach by establishing the essential role in apicoplast protein import for a new factor with intriguing localization and evolutionary history.
PMCID: PMC3228799  PMID: 22144892
11.  Screening and control of methicillin-resistant Staphylococcus aureus in 186 intensive care units: different situations and individual solutions 
Critical Care  2011;15(6):R285.
Controversy exists about the benefit of screening for prevention of methicillin-resistant Staphylococcus aureus (MRSA) in intensive care units (ICUs) and recent studies have shown conflicting results. The aim of this observational study was to describe and evaluate the association between MRSA incidence densities (IDs) and screening and control measures in ICUs participating in the German Nosocomial Infection Surveillance System.
The surveillance module for multidrug-resistant bacteria collects data on MRSA cases in ICUs with the aim to provide a national reference and a tool for evaluation of infection control management. The median IDs of MRSA cases per 1000 patient-days (pd) with the interquartile range (IQR) were calculated from the pooled data of 186 ICUs and correlated with parameters derived from a detailed questionnaire regarding ICU structure, microbiological diagnostics and MRSA screening and control measures. The association between questionnaire results and MRSA cases was evaluated by generalized linear regression models.
One hundred eighty-six ICUs submitted data on MRSA cases for 2007 and 2008 and completed the questionnaire. During the period of analysis, 4935 MRSA cases occurred in these ICUs; of these, 3928 (79.6%) were imported and 1007 MRSA cases (20.4%) were ICU-acquired. Median MRSA IDs were 3.23 (IQR 1.24-5.73), 2.24 (IQR 0.63-4.30) and 0.64 (IQR 0.17-1.39) per 1000 pd for all cases, imported and ICU-acquired MRSA cases, respectively. MRSA IDs as well as implemented MRSA screening and control measures varied widely between ICUs. ICUs performing universal admission screening had significantly higher MRSA IDs than ICUs performing targeted or no screening. Separate regression models for ICUs with different screening strategies included the incidence of imported MRSA cases, the type of ICU, and the length of stay in independent association with the number of ICU-acquired MRSA cases.
The analysis shows that MRSA IDs and structural parameters differ considerably between ICUs. In response, ICUs have combined screening and control measures in many ways to achieve various individual solutions. The incidence of imported MRSA cases might be helpful for consideration in the planning of MRSA control programmes.
PMCID: PMC3388634  PMID: 22118016
12.  T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest 
PLoS ONE  2010;5(11):e14057.
Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.
PMCID: PMC2989910  PMID: 21124925
13.  A novel multifunctional oligonucleotide microarray for Toxoplasma gondii 
BMC Genomics  2010;11:603.
Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators.
Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform future array designs.
In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.
PMCID: PMC3017859  PMID: 20974003
14.  Coordinated Progression through Two Subtranscriptomes Underlies the Tachyzoite Cycle of Toxoplasma gondii 
PLoS ONE  2010;5(8):e12354.
Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission.
Methodology/Principal Findings
Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities.
Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.
PMCID: PMC2928733  PMID: 20865045
15.  Phenotypic and Gene Expression Changes among Clonal Type I Strains of Toxoplasma gondii▿ †  
Eukaryotic Cell  2009;8(12):1828-1836.
Toxoplasma gondii has an unusual population structure consisting of three clonal lineages that predominate in North America and Europe. This simple pattern has encouraged the use of only a few laboratory isolates that are representative of each lineage. Principle among these is the type I RH strain, originally isolated from a child with encephalitis some 70 years ago. Comparison of different passages of the RH strain that have been propagated differently over the intervening time period revealed that the commonly used clonal line called RH-ERP was not representative of natural isolates of the type I lineage. Notably, RH-ERP formed much larger plaques than other type 1 strains, including a separate, earlier derived isolate of the RH strain. The RH-ERP variant also showed enhanced extracellular survival, faster growth, and decreased differentiation compared to the prototype type I strain GT1. Comparison of gene expression differences in the RH-ERP line revealed that several ABC transporters were upregulated, which may provide a growth advantage in vitro. These findings illustrate that dramatic phenotypic changes can arise in laboratory strains, emphasizing the need for comparison with recent clinical isolates.
PMCID: PMC2794221  PMID: 19801420
16.  The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements 
Molecular Microbiology  2008;68(6):1502-1518.
Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6–8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a ‘poised’ chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.
PMCID: PMC2440561  PMID: 18433450
17.  Risk factors for the development of nosocomial pneumonia and mortality on intensive care units: application of competing risks models 
Critical Care  2008;12(2):R44.
Pneumonia is a very common nosocomial infection in intensive care units (ICUs). Many studies have investigated risk factors for the development of infection and its consequences. However, the evaluation in most of theses studies disregards the fact that there are additional competing events, such as discharge or death.
A prospective cohort study was conducted over 18 months in five intensive care units at one university hospital. All patients that were admitted for at least 2 days were included, and surveillance of nosocomial pneumonia was conducted. Various potential risk factors (baseline- and time-dependent) were evaluated in two competing risks models: the acquisition of nosocomial pneumonia and discharge (dead or alive; model 1) and for the risk of death in the ICU and discharge alive (model 2).
Patients from 1,876 admissions were included. A total of 158 patients developed nosocomial pneumonia. The main risk factors for nosocomial pneumonia in the multivariate analysis in model 1 were: elective surgery (cause-specific hazard ratio = 1.95; 95% CI 1.33 to 2.85) or emergency surgery (1.59; 95% CI 1.10 to 2.28) prior to ICU admission, usage of a nasogastric tube (3.04; 95% CI 1.25 to 7.37) and mechanical ventilation (5.90; 95% CI 2.47 to 14.09). Nosocomial pneumonia prolonged the length of ICU stay but was not directly associated with a fatal outcome (p = 0.55).
More studies using competing risk models, which provide more accurate data compared to naive survival curves or logistic models, should be carried out to verify the impact of risk factors and patient characteristics for the acquisition of nosocomial infections and infection-associated mortality.
PMCID: PMC2447589  PMID: 18384672
18.  Changes in the Expression of Human Cell Division Autoantigen-1 Influence Toxoplasma gondii Growth and Development 
PLoS Pathogens  2006;2(10):e105.
Toxoplasma is a significant opportunistic pathogen in AIDS, and bradyzoite differentiation is the critical step in the pathogenesis of chronic infection. Bradyzoite development has an apparent tropism for cells and tissues of the central nervous system, suggesting the need for a specific molecular environment in the host cell, but it is unknown whether this environment is parasite directed or the result of molecular features specific to the host cell itself. We have determined that a trisubstituted pyrrole acts directly on human and murine host cells to slow tachyzoite replication and induce bradyzoite-specific gene expression in type II and III strain parasites but not type I strains. New mRNA synthesis in the host cell was required and indicates that novel host transcripts encode signals that were able to induce parasite development. We have applied multivariate microarray analyses to identify and correlate host gene expression with specific parasite phenotypes. Human cell division autoantigen-1 (CDA1) was identified in this analysis, and small interfering RNA knockdown of this gene demonstrated that CDA1 expression causes the inhibition of parasite replication that leads subsequently to the induction of bradyzoite differentiation. Overexpression of CDA1 alone was able to slow parasite growth and induce the expression of bradyzoite-specific proteins, and thus these results demonstrate that changes in host cell transcription can directly influence the molecular environment to enable bradyzoite development. Investigation of host biochemical pathways with respect to variation in strain type response will help provide an understanding of the link(s) between the molecular environment in the host cell and parasite development.
Toxoplasma is a common opportunistic pathogen among immunocompromised populations that include subjects undergoing organ transplant, the fetus during early gestation, and persons with AIDS. The parasite escapes the host immune system by forming a dormant tissue cyst, and this chronic infection, as well as the clinical manifestation of disease, is observed primarily in cells and tissues of the brain and eye. Although it is not yet understood how the disease state is established, in this study researchers demonstrate that Toxoplasma can take cues from specific changes in host cell gene expression to initiate switching to the tissue cyst, and they discover that a single gene, designated human cell division autoantigen-1 (CDA1), is able to impose significant influence on the course of infection and cyst development. These studies are the first to identify a host gene that links the molecular environment in the cell to parasite development. It is interesting that the response to the host cell is not uniform among parasite strains, as acutely virulent strains appear to ignore the host and continue to proliferate until the cell is destroyed.
PMCID: PMC1626100  PMID: 17069459
19.  The transcriptome of Toxoplasma gondii 
BMC Biology  2005;3:26.
Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown.
We have used serial analysis of gene expression (SAGE) to define the Toxoplasma gondii transcriptome of the intermediate-host life cycle that leads to the formation of the bradyzoite/tissue cyst. A broad view of gene expression is provided by >4-fold coverage from nine distinct libraries (~300,000 SAGE tags) representing key developmental transitions in primary parasite populations and in laboratory strains representing the three canonical genotypes. SAGE tags, and their corresponding mRNAs, were analyzed with respect to abundance, uniqueness, and antisense/sense polarity and chromosome distribution and developmental specificity.
This study demonstrates that phenotypic transitions during parasite development were marked by unique stage-specific mRNAs that accounted for 18% of the total SAGE tags and varied from 1–5% of the tags in each developmental stage. We have also found that Toxoplasma mRNA pools have a unique parasite-specific composition with 1 in 5 transcripts encoding Apicomplexa-specific genes functioning in parasite invasion and transmission. Developmentally co-regulated genes were dispersed across all Toxoplasma chromosomes, as were tags representing each abundance class, and a variety of biochemical pathways indicating that trans-acting mechanisms likely control gene expression in this parasite. We observed distinct similarities in the specificity and expression levels of mRNAs in primary populations (Day-6 post-sporozoite infection) that occur prior to the onset of bradyzoite development that were uniquely shared with the virulent Type I-RH laboratory strain suggesting that development of RH may be arrested. By contrast, strains from Type II-Me49B7 and Type III-VEGmsj contain SAGE tags corresponding to bradyzoite genes, which suggests that priming of developmental expression likely plays a role in the greater capacity of these strains to complete bradyzoite development.
PMCID: PMC1325263  PMID: 16324218

Results 1-19 (19)