The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi

We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N(5)-formyl-N(5)-hydroxyornithine, serine, N(5)-hydroxyornithine, and N(5)-acyl-N(5)-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence

Phenotypic and Transcriptomic Response of Auxotrophic Mycobacterium avium Subsp. paratuberculosis leuD Mutant under Environmental Stress

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP

Two Origins for the Gene Encoding α-Isopropylmalate Synthase in Fungi

BACKGROUND: The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy. METHODOLOGY/PRINCIPAL FINDINGS: Here we identified the leuA gene encoding alpha-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently. CONCLUSIONS/SIGNIFICANCE: The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events

Towards a competency-based doctoral curriculum at the University of Zambia: lessons from practice

We describe a collaborative, iterative, and participatory process that we undertook to develop and adopt a competency-based doctoral curriculum framework at the University of Zambia. There needs to be more than the traditional unstructured apprenticeship of PhD training in a knowledge-based economy where PhD graduates are expected to contribute to industry problem-solving. The lack of industry-driven competencies and, to some extent, limited skills possessed by PhD graduates relative to the demands of employers has led to the misclassification of doctoral degrees as mere paper certificates. Further, under traditional PhD training without specific core competencies, it has led to criticisms of such PhD studies as a waste of resources. The calls to rethink doctoral development in broader employment contexts led many countries to redesign their PhD programs. Training has increasingly introduced industrial linkages and industry-defined research projects to increase the attractiveness of doctoral students. Whereas developed countries have made significant reforms towards competency-based PhD training, little or nothing has been done in developing countries, especially in sub-Saharan Africa. This against the demands that Africa needs more than 100,000 PhDs in the next decade to spur economic development. Against this background, the University of Zambia has developed an industry-driven structured competency-based PhD curriculum framework. The framework will guide and support the development of standardized program-specific PhD curricula, delivery, and assessment of competencies at the University of Zambia, ensuring that doctoral students acquire skills and demonstrate core competencies that are transferable and applicable in industry settings. This framework focuses on the development of specific competencies that are necessary for successful PhD completion. The competencies are divided into three main categories: research, teaching, and professional development. Each category is then broken down into ten core competencies from which respective doctoral programs will develop sub-competencies. It is from these core competencies and sub-competencies that learning outcomes, assessment methods, and teaching activities are developed. It is envisioned that this new competency-based doctoral curriculum framework will be a helpful tool in training a cadre of professionals and researchers who benefit the industry and contribute to economic and societal development

Priming with a Recombinant Pantothenate Auxotroph of Mycobacterium bovis BCG and Boosting with MVA Elicits HIV-1 Gag Specific CD8+ T Cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8+ T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4+ T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge

The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi

Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.Peer reviewedVeterinary Pathobiolog