Mutation at the "exit gate" of the salmonella gyrase a subunit suppresses a defect in the gyrase B subunit

In Salmonella enterica serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele gyrB651) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the gyrA gene (allele gyrA659), a T467S substitution, which partially suppresses the supercoiling defect of gyrB651. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a gyrA mutation that compensates for a gyrB defect.     

Mycobacterium marinum MgtC Plays a Role in Phagocytosis but is Dispensable for Intracellular Multiplication

MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg²⁺ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg²⁺ deprivation and is required for optimal growth in Mg²⁺-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg²⁺ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen''s lifestyles.     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.     

From dissimilarities among species to dissimilarities among communities: a double principal coordinate analysis

This paper proposes a new method to reverse engineer gene regulatory networks from experimental data. The modeling framework used is time-discrete deterministic dynamical systems, with a finite set of states for each of the variables. The simplest examples of such models are Boolean networks, in which variables have only two possible states. The use of a larger number of possible states allows a finer discretization of experimental data and more than one possible mode of action for the variables, depending on threshold values. Furthermore, with a suitable choice of state set, one can employ powerful tools from computational algebra, that underlie the reverse-engineering algorithm, avoiding costly enumeration strategies. To perform well, the algorithm requires wildtype together with perturbation time courses. This makes it suitable for small to meso-scale networks rather than networks on a genome-wide scale. An analysis of the complexity of the algorithm is performed. The algorithm is validated on a recently published Boolean network model of segment polarity development in Drosophila melanogaster.     

Consensus genetic structuring and typological value of markers using multiple co-inertia analysis

Working with weakly congruent markers means that consensus genetic structuring of populations requires methods explicitly devoted to this purpose. The method, which is presented here, belongs to the multivariate analyses. This method consists of different steps. First, single-marker analyses were performed using a version of principal component analysis, which is designed for allelic frequencies (%PCA). Drawing confidence ellipses around the population positions enhances %PCA plots. Second, a multiple co-inertia analysis (MCOA) was performed, which reveals the common features of single-marker analyses, builds a reference structure and makes it possible to compare single-marker structures with this reference through graphical tools. Finally, a typological value is provided for each marker. The typological value measures the efficiency of a marker to structure populations in the same way as other markers. In this study, we evaluate the interest and the efficiency of this method applied to a European and African bovine microsatellite data set. The typological value differs among markers, indicating that some markers are more efficient in displaying a consensus typology than others. Moreover, efficient markers in one collection of populations do not remain efficient in others. The number of markers used in a study is not a sufficient criterion to judge its reliability. "Quantity is not quality".     

Dual role of the MgtC virulence factor in host and non-host environments

MgtC is required for intramacrophage replication of intracellular pathogens and growth in low Mg(2+) medium. A link between these two phenotypes has been proposed due to putative Mg(2+) deprivation inside phagosome. MgtC is part of a family of proteins that share a conserved N-terminal transmembrane domain and a variable C-terminal domain. A combination of predictive and experimental approaches indicates that the Salmonella MgtC C-terminal domain is cytoplasmic, adopts a fold also found in metal transporters and RNA interacting domain, and does not bind Mg(2+). MgtC homologues from diverse gamma-proteobacteria, including the extracellular pathogens Yersinia pestis, Photorhabdus luminescens and Pseudomonas aeruginosa, have been expressed in a SalmonellaDeltamgtC strain. The Y. pestis MgtC fully replaced the Salmonella MgtC whereas P. luminescens or P. aeruginosa MgtC complemented only in low Mg(2+) medium, thus dissociating for the first time the two MgtC-related phenotypes. In addition, we identified single amino acids changes that prevent or promote MgtC role in macrophages without affecting MgtC role in low Mg(2+) culture. A SalmonellaDeltamgtC strain showed elongated and autoaggregated bacteria in low Mg(2+) medium but not in macrophages. Taken together our results suggest that MgtC has a dual role when bacteria localize in macrophages or low Mg(2+) environment.     

MgtC: a key player in intramacrophage survival

Several bacterial pathogens have evolved strategies to survive in macrophages and create a replicative niche within phagosomes. The bacterial factor MgtC is a key player in intramacrophage survival, being important for virulence in diverse intracellular pathogens. MgtC is also required for growth under magnesium limitation. Recent studies provide new clues on the role of MgtC in macrophages, which seems to be unlinked to adaptation to a low Mg(2+) microenvironment. In addition, we discuss the unexpected finding that MgtC modulates host P-type ATPase activity.     

Design and evaluation of a novel series of 2,3-oxidosqualene cyclase inhibitors with low systemic exposure, relationship between pharmacokinetic properties and ocular toxicity

We describe the discovery of novel potent inhibitors of 2,3-oxidosqualene:lanosterol cyclase inhibitors (OSCi) from a focused pharmacophore-based screen. Optimization of the most tractable hits gave a series of compounds showing inhibition of cholesterol biosynthesis at 2mg/kg in the rat with distinct pharmacokinetic profiles. Two compounds were selected for toxicological study in the rat for 21 days in order to test the hypothesis that low systemic exposure could be used as a strategy to avoid the ocular side effects previously described with OSCi. We demonstrate that for this series of inhibitors, a reduction of systemic exposure is not sufficient to circumvent cataract liabilities.     

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg²⁺ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg²⁺ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.