Lance E. Palmer

YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNF-alpha production and downregulation of the MAP kinases p38 and JNK.

Exposure of macrophages to lipopolysaccharide (LPS) leads to production of the pro‐inflammatory cytokine, tumour necrosis factor alpha (TNF‐α). Previous studies have suggested that pathogenic Yersinia spp. inhibit LPS‐mediated production of TNF‐α in macrophages, and that one of the Yop proteins secreted by the plasmid‐encoded type III pathway is required for this activity. We found that TNF‐α production was inhibited when J774A.1 murine macrophages were infected with wild‐type Y. pseudotuberculosis but not with an isogenic ysc mutant defective for Yop secretion. We inactivated multiple yop genes to identify which of these factors are required for the inhibition of TNF‐α production. A mutant unable to express yopJ was defective for the inhibition of TNF‐α production. Production of TNF‐α is regulated at the transcriptional and translational levels by several mitogen‐activated protein (MAP) kinases. The MAP kinases p38 and JNK underwent sustained activation in macrophages infected with the yopJ mutant. Conversely, p38 and JNK were downregulated in macrophages infected with the wild‐type strain. The ability of the yopJ mutant to downregulate p38 and JNK and to inhibit production of TNF‐α was restored by the expression of yopJ+in trans. Therefore, YopJ is required for Y. pseudotuberculosis to downregulate MAP kinases and inhibit the production of TNF‐α in macrophages.

Disruption of contactin 4 in three subjects with autism spectrum disorder

Background: Autism spectrum disorder (ASD) is a developmental disorder of the central nervous system of largely unknown aetiology. The prevalence of the syndrome underscores the need for biological markers and a clearer understanding of pathogenesis. For these reasons, a genetic study of idiopathic ASD was undertaken. Methods and results: Array based comparative genomic hybridisation identified a paternally inherited chromosome 3 copy number variation (CNV) in three subjects: a deletion in two siblings and a duplication in a third, unrelated individual. These variations were fluorescence in situ hybridisation (FISH) validated and the end points further delineated using a custom fine tiling oligonucleotide array. Polymerase chain reaction (PCR) products unique to the rearrangements were amplified and sequence analysis revealed the variations to have resulted from Alu Y mediated unequal recombinations interrupting contactin 4 (CNTN4). Conclusion: CNTN4 plays an essential role in the formation, maintenance, and plasticity of neuronal networks. Disruption of this gene is known to cause developmental delay and mental retardation. This report suggests that mutations affecting CNTN4 function may be relevant to ASD pathogenesis.

An Improved Vaccine for Prevention of Respiratory Tularemia Caused by Francisella tularensis SchuS4 Strain

Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB(Ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB(Ft) mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB(Ft) vaccinated group as compared to the LVS vaccinated mice. sodB(Ft) vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB(Ft) vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB(Ft) mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS.

Global Gene Expression Profiling of Yersinia pestis Replicating inside Macrophages Reveals the Roles of a Putative Stress-Induced Operon in Regulating Type III Secretion and Intracellular Cell Division

ABSTRACT Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Δy2313-y2316 or ΔorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment.

A Transposon Site Hybridization Screen Identifies galU and wecBC as Important for Survival of Yersinia pestis in Murine Macrophages

Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes that are important for Y. pestis survival in macrophages, a library comprised of ∼31,500 Y. pestis KIM6+ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-d-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6+ galU mutant and a KIM6+ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6+ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6+, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.

FeoB-mediated uptake of iron by Francisella tularensis.

ABSTRACT Francisella tularensis, the bacterial cause of tularemia, infects the liver and replicates in hepatocytes in vivo and in vitro. However, the factors that govern adaptation of F. tularensis to the intrahepatocytic niche have not been identified. Using cDNA microarrays, we determined the transcriptional profile of the live vaccine strain (LVS) of F. tularensis grown in the FL83B murine hepatocytic cell line compared to that of F. tularensis cultured in broth. The fslC gene of the fsl operon was the most highly upregulated. Deletion of fslC eliminated the ability of the LVS to produce siderophore, which is involved in uptake of ferric iron, but it did not impair its growth in hepatocytes, A549 epithelial cells, or macrophages. Therefore, we sought an alternative means by which F. tularensis might obtain iron. Deletion of feoB, which encodes a putative ferrous iron transporter, retarded replication of the LVS in iron-restricted media, reduced its growth in hepatocytic and epithelial cells, and impaired its acquisition of iron. Survival of mice infected intradermally with a lethal dose of the LVS was slightly improved by deletion of fslC but was not altered by loss of feoB. However, the ΔfeoB mutant showed diminished ability to colonize the lungs, liver, and spleen of mice that received sublethal inocula. Thus, FeoB represents a previously unidentified mechanism for uptake of iron by F. tularensis. Moreover, failure to produce a mutant strain lacking both feoB and fslC suggests that FeoB and the proteins of the fsl operon are the only major means by which F. tularensis acquires iron.

IFN-γ Alters the Response of Borrelia burgdorferi-Activated Endothelium to Favor Chronic Inflammation

Borrelia burgdorferi, the agent of Lyme disease, promotes proinflammatory changes in the endothelium that lead to the recruitment of leukocytes. The host immune response to infection results in increased levels of IFN-γ in the serum and lesions of Lyme disease patients that correlate with greater severity of disease. Therefore, the effect of IFN-γ on the gene expression profile of primary human endothelial cells exposed to B. burgdorferi was determined. B. burgdorferi and IFN-γ synergistically augmented the expression of 34 genes, 7 of which encode chemokines. Six of these (CCL7, CCL8, CX3CL1, CXCL9, CXCL10, and CXCL11) attract T lymphocytes, and one (CXCL2) is specific for neutrophils. Synergistic production of the attractants for T cells was confirmed at the protein level. IL-1β, TNF-α, and LPS also cooperated with IFN-γ to induce synergistic production of CXCL10 by the endothelium, indicating that IFN-γ potentiates inflammation in concert with a variety of mediators. An in vitro model of the blood vessel wall revealed that an increased number of human T lymphocytes traversed the endothelium exposed to B. burgdorferi and IFN-γ, as compared with unstimulated endothelial monolayers. In contrast, addition of IFN-γ diminished the migration of neutrophils across the B. burgdorferi-activated endothelium. IFN-γ thus alters gene expression by endothelia exposed to B. burgdorferi in a manner that promotes recruitment of T cells and suppresses that of neutrophils. This modulation may facilitate the development of chronic inflammatory lesions in Lyme disease.

On the importance of being finished

The publication of an increasing number of draft genome sequences presents problems that will only be resolved by improved search tools and by complete finishing of the sequences - and their deposition in publicly accessible databases.