Acute and Persistent Mycobacterium tuberculosis Infections Depend on the Thiol Peroxidase TPX

The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H2O2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-γ activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2) knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models

Dormancy within Staphylococcus epidermidis biofilms : a transcriptomic analysis by RNA-seq

The proportion of dormant bacteria within Staphylococcus epidermidis biofilms may determine its inflammatory profile. Previously, we have shown that S. epidermidis biofilms with higher proportions of dormant bacteria have reduced activation of murine macrophages. RNA-sequencing was used to identify the major transcriptomic differences between S. epidermidis biofilms with different proportions of dormant bacteria. To accomplish this goal, we used an in vitro model where magnesium allowed modulation of the proportion of dormant bacteria within S. epidermidis biofilms. Significant differences were found in the expression of 147 genes. A detailed analysis of the results was performed based on direct and functional gene interactions. Biological processes among the differentially expressed genes were mainly related to oxidation-reduction processes and acetyl-CoA metabolic processes. Gene set enrichment revealed that the translation process is related to the proportion of dormant bacteria. Transcription of mRNAs involved in oxidation-reduction processes was associated with higher proportions of dormant bacteria within S. epidermidis biofilm. Moreover, the pH of the culture medium did not change after the addition of magnesium, and genes related to magnesium transport did not seem to impact entrance of bacterial cells into dormancy.The authors thank Stephen Lorry at Harvard Medical School for providing CLC Genomics software. This work was funded by Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009, FCOMP-01-0124-FEDER-014309, FCOMP-01-0124-FEDER-022718 (FCT PEst-C/SAU/LA0002/2011), QOPNA research unit (project PEst-C/QUI/UI0062/2011), and CENTRO-07-ST24-FEDER-002034. The following authors had an individual FCT fellowship: VC (SFRH/BD/78235/2011) and AF (2SFRH/BD/62359/2009)

Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival

Genome-Wide Screen for Mycobacterium tuberculosis Genes That Regulate Host Immunity

In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNFα, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence

Trost E, Ott L, Schneider J, et al. The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence. BMC Genomics. 2010;11(1): 728