Limited Transcriptional Responses of Rickettsia rickettsii Exposed to Environmental Stimuli

Rickettsiae are strict obligate intracellular pathogens that alternate between arthropod and mammalian hosts in a zoonotic cycle. Typically, pathogenic bacteria that cycle between environmental sources and mammalian hosts adapt to the respective environments by coordinately regulating gene expression such that genes essential for survival and virulence are expressed only upon infection of mammals. Temperature is a common environmental signal for upregulation of virulence gene expression although other factors may also play a role. We examined the transcriptional responses of Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, to a variety of environmental signals expected to be encountered during its life cycle. R. rickettsii exposed to differences in growth temperature (25°C vs. 37°C), iron limitation, and host cell species displayed nominal changes in gene expression under any of these conditions with only 0, 5, or 7 genes, respectively, changing more than 3-fold in expression levels. R. rickettsii is not totally devoid of ability to respond to temperature shifts as cold shock (37°C vs. 4°C) induced a change greater than 3-fold in up to 56 genes. Rickettsiae continuously occupy a relatively stable environment which is the cytosol of eukaryotic cells. Because of their obligate intracellular character, rickettsiae are believed to be undergoing reductive evolution to a minimal genome. We propose that their relatively constant environmental niche has led to a minimal requirement for R. rickettsii to respond to environmental changes with a consequent deletion of non-essential transcriptional response regulators. A minimal number of predicted transcriptional regulators in the R. rickettsii genome is consistent with this hypothesis

miR-182 and miR-10a Are Key Regulators of Treg Specialisation and Stability during Schistosome and Leishmania-associated Inflammation

A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4+Foxp3+ regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA “regulatory hubs” miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4+Foxp3+ cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function

Simultaneous Host-Pathogen Transcriptome Analysis during Granulibacter bethesdensis Infection of Neutrophils from Healthy Subjects and Patients with Chronic Granulomatous Disease

Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species (ROS) due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by PMN of CGD patients (CGD PMN) and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in PMN from healthy subjects (normal PMN) and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of antiapoptotic genes (e.g., XIAP and GADD45B) and downregulation of proapoptotic genes (e.g., CASP8 and APAF1) in infected PMN. Transcript and protein levels of inflammation- and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered the expression of ROS resistance genes in the presence of normal but not CGD PMN. Levels of bacterial stress response genes, including the ClpB gene, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knockdown demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included the upregulation of pyruvate dehydrogenase. Pharmacological inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis of Granulibacter in cells from permissive (CGD) and nonpermissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen.This is the publisher’s final pdf. The published article is copyrighted by American Society for Microbiology and can be found at: http://iai.asm.org

Comparative Pathogenesis of Three Human and Zoonotic SARS-CoV Strains in Cynomolgus Macaques

The severe acute respiratory syndrome (SARS) epidemic was characterized by increased pathogenicity in the elderly due to an early exacerbated innate host response. SARS-CoV is a zoonotic pathogen that entered the human population through an intermediate host like the palm civet. To prevent future introductions of zoonotic SARS-CoV strains and subsequent transmission into the human population, heterologous disease models are needed to test the efficacy of vaccines and therapeutics against both late human and zoonotic isolates. Here we show that both human and zoonotic SARS-CoV strains can infect cynomolgus macaques and resulted in radiological as well as histopathological changes similar to those seen in mild human cases. Viral replication was higher in animals infected with a late human phase isolate compared to a zoonotic isolate. While there were significant differences in the number of host genes differentially regulated during the host responses between the three SARS-CoV strains, the top pathways and functions were similar and only apparent early during infection with the majority of genes associated with interferon signaling pathways. This study characterizes critical disease models in the evaluation and licensure of therapeutic strategies against SARS-CoV for human use

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence

In Vivo Interactions of Ecotropic and Polytropic Murine Leukemia Viruses in Mixed Retrovirus Infections

Mixed retrovirus infections are the rule rather than the exception in mice and other species, including humans. Interactions of retroviruses in mixed infections and their effects on disease induction are poorly understood. Upon infection of mice, ecotropic retroviruses recombine with endogenous proviruses to generate polytropic viruses that utilize different cellular receptors. Interactions among the retroviruses of this mixed infection facilitate disease induction. Using mice infected with defined mixtures of the ecotropic Friend murine leukemia virus (F-MuLV) and different polytropic viruses, we demonstrate several dramatic effects of mixed infections. Remarkably, inoculation of F-MuLV with polytropic MuLVs completely suppressed the generation of new recombinant viruses and dramatically altered disease induction. Coinoculation of F-MuLV with one polytropic virus significantly lengthened survival times, while inoculation with another polytropic MuLV induced a rapid and severe neurological disease. In both instances, the level of the polytropic MuLV was increased 100- to 1,000-fold, whereas the ecotropic MuLV level remained unchanged. Surprisingly, nearly all of the polytropic MuLV genomes were packaged within F-MuLV virions (pseudotyped) very soon after infection. At this time, only a fractional percentage of cells in the mouse were infected by either virus, indicating that the coinoculated viruses had infected the same small subpopulation of susceptible cells. The profound amplification of polytropic MuLVs in coinfected mice may be facilitated by pseudotyping or, alternatively, by transactivation of the polytropic virus in the coinfected cells. This study illustrates the complexity of the interactions between components of mixed retrovirus infections and the dramatic effects of these interactions on disease processes

Genetic Diversity of the Q Fever Agent, Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

Coxiella burnetii, a gram-negative obligate intracellular bacterium, causes human Q fever and is considered a potential agent of bioterrorism. Distinct genomic groups of C. burnetii are revealed by restriction fragment-length polymorphisms (RFLP). Here we comprehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-grouped and four ungrouped isolates from disparate sources to a high-density custom Affymetrix GeneChip containing all open reading frames (ORFs) of the Nine Mile phase I (NMI) reference isolate. We confirmed the relatedness of RFLP-grouped isolates and showed that two ungrouped isolates represent distinct genomic groups. Isolates contained up to 20 genomic polymorphisms consisting of 1 to 18 ORFs each. These were mostly complete ORF deletions, although partial deletions, point mutations, and insertions were also identified. A total of 139 chromosomal and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI coding capacity. Approximately 67% of all deleted ORFs were hypothetical, while 9% were annotated in NMI as nonfunctional (e.g., frameshifted). The remaining deleted ORFs were associated with diverse cellular functions. The only deletions associated with isogenic NMI variants of attenuated virulence were previously described large deletions containing genes involved in lipopolysaccharide (LPS) biosynthesis, suggesting that these polymorphisms alone are responsible for the lower virulence of these variants. Interestingly, a variant of the Australia QD isolate producing truncated LPS had no detectable deletions, indicating LPS truncation can occur via small genetic changes. Our results provide new insight into the genetic diversity and virulence potential of Coxiella species

Growth kinetics of <i>R. rickettsii</i> under different experimental conditions.

<p>A) Growth of <i>R. rickettsii</i> in Vero cells incubated at either 34°C or 25°C. B) Growth of <i>R. rickettsii</i> at 37°C in ISE6 and Vero cells. C) Growth of <i>R. rickettsii</i> in ISE6 cells at 22°C.</p

Genomic Comparison of Virulent Rickettsia rickettsii Sheila Smith and Avirulent Rickettsia rickettsii Iowa▿

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. To identify genes involved in the virulence of R. rickettsii, the genome of an avirulent strain, R. rickettsii Iowa, was sequenced and compared to the genome of the virulent strain R. rickettsii Sheila Smith. R. rickettsii Iowa is avirulent in a guinea pig model of infection and displays altered plaque morphology with decreased lysis of infected host cells. Comparison of the two genomes revealed that R. rickettsii Iowa and R. rickettsii Sheila Smith share a high degree of sequence identity. A whole-genome alignment comparing R. rickettsii Iowa to R. rickettsii Sheila Smith revealed a total of 143 deletions for the two strains. A subsequent single-nucleotide polymorphism (SNP) analysis comparing Iowa to Sheila Smith revealed 492 SNPs for the two genomes. One of the deletions in R. rickettsii Iowa truncates rompA, encoding a major surface antigen (rickettsial outer membrane protein A [rOmpA]) and member of the autotransporter family, 660 bp from the start of translation. Immunoblotting and immunofluorescence confirmed the absence of rOmpA from R. rickettsii Iowa. In addition, R. rickettsii Iowa is defective in the processing of rOmpB, an autotransporter and also a major surface antigen of spotted fever group rickettsiae. Disruption of rompA and the defect in rOmpB processing are most likely factors that contribute to the avirulence of R. rickettsii Iowa. Genomic differences between the two strains do not significantly alter gene expression as analysis of microarrays revealed only four differences in gene expression between R. rickettsii Iowa and R. rickettsii strain R. Although R. rickettsii Iowa does not cause apparent disease, infection of guinea pigs with this strain confers protection against subsequent challenge with the virulent strain R. rickettsii Sheila Smith