Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium's ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes

Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis <it>in vivo </it>unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that <it>chronic </it>HIV-<it>replication in non-endothelial cells </it>may produce novel factors that provoke angiogenic pathways.</p> <p>Methods</p> <p>Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth <it>in vitro </it>over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses.</p> <p>Results</p> <p>By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development.</p> <p>Conclusion</p> <p>Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p = 10^-4to 10^-12). Bioinformatics analyses suggest that overproduction of PTKs and other kinases in HIV-infected cells has <it>suppressed </it>VEGF/VEGFR-PTK expression and promoted <it>VEGFR-independent </it>pathways. This unique mechanism is similar to that observed in neovascularization and angiogenesis during embryogenesis. Validation of clinically relevant proteins by gene-silencing and translational studies <it>in vivo </it>would identify specific targets that can be used for early diagnosis of angiogenic disorders and future development of inhibitors of angiopathies. This is the first comprehensive study to demonstrate that HIV-infection alone, without any co-infection or treatment, can induce numerous "embryonic" proteins and kinases capable of generating novel <it>VEGF-independent </it>angiogenic pathways.</p

Interactions between the microbiota and pathogenic bacteria in the gut

The microbiome has an important role in human health. Changes in the microbiota can confer resistance to or promote infection by pathogenic bacteria. Antibiotics have a profound impact on the microbiota that alters the nutritional landscape of the gut and can lead to the expansion of pathogenic populations. Pathogenic bacteria exploit microbiota-derived sources of carbon and nitrogen as nutrients and regulatory signals to promote their own growth and virulence. By eliciting inflammation, these bacteria alter the intestinal environment and use unique systems for respiration and metal acquisition to drive their expansion. Unravelling the interactions between the microbiota, the host and pathogenic bacteria will produce strategies for manipulating the microbiota against infectious diseases