A Putative P-Type ATPase Required for Virulence and Resistance to Haem Toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem

Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance

High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials

Chikungunya Disease: Infection-Associated Markers from the Acute to the Chronic Phase of Arbovirus-Induced Arthralgia

At the end of 2005, an outbreak of fever associated with joint pain occurred in La Réunion. The causal agent, chikungunya virus (CHIKV), has been known for 50 years and could thus be readily identified. This arbovirus is present worldwide, particularly in India, but also in Europe, with new variants returning to Africa. In humans, it causes a disease characterized by a typical acute infection, sometimes followed by persistent arthralgia and myalgia lasting months or years. Investigations in the La Réunion cohort and studies in a macaque model of chikungunya implicated monocytes-macrophages in viral persistence. In this Review, we consider the relationship between CHIKV and the immune response and discuss predictive factors for chronic arthralgia and myalgia by providing an overview of current knowledge on chikungunya pathogenesis. Comparisons of data from animal models of the acute and chronic phases of infection, and data from clinical series, provide information about the mechanisms of CHIKV infection–associated inflammation, viral persistence in monocytes-macrophages, and their link to chronic signs

Functional Genetic Diversity among Mycobacterium tuberculosis Complex Clinical Isolates: Delineation of Conserved Core and Lineage-Specific Transcriptomes during Intracellular Survival

Tuberculosis exerts a tremendous burden on global health, with ∼9 million new infections and ∼2 million deaths annually. The Mycobacterium tuberculosis complex (MTC) was initially regarded as a highly homogeneous population; however, recent data suggest the causative agents of tuberculosis are more genetically and functionally diverse than appreciated previously. The impact of this natural variation on the virulence and clinical manifestations of the pathogen remains largely unknown. This report examines the effect of genetic diversity among MTC clinical isolates on global gene expression and survival within macrophages. We discovered lineage-specific transcription patterns in vitro and distinct intracellular growth profiles associated with specific responses to host-derived environmental cues. Strain comparisons also facilitated delineation of a core intracellular transcriptome, including genes with highly conserved regulation across the global panel of clinical isolates. This study affords new insights into the genetic information that M. tuberculosis has conserved under selective pressure during its long-term interactions with its human host

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

The remarkable capacity for gut microbial and host interactions

The stunning complexity of the resident microbiota and the intricate pathways of microbial and host interactions provide a massive adaptive capacity for mammals. In this addendum we reflect on our recent publication on Toll-like receptor 2 deficiency related colonic mucosal epigenetic, immunologic and microbiomic changes. Our findings underscored the tremendous flexibility of the gut and its microbiota. This flexibility can provide means to overcome significant environmental or genetic challenges. In the meantime, the challenged intestinal system may become vulnerable to otherwise tolerable insults. In such instances, the fine-tuned mutualistic balance between the gut and its microflora may collapse leading to dysbiosis and disease. The ultimate challenge for biomedical research in these cases is to find optimal means for the restoration and maintenance of healthy host physiology