Conversion of commensal Escherichia coli K-12 to an invasive form via expression of a mutant histone-like protein.

UNLABELLED: The HUα(E38K, V42L) mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397-16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUα(E38K, V42L) was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins Bim(EL) and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUα(E38K, V42L) variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes. IMPORTANCE: Escherichia coli K-12 is well established as a benign laboratory strain and a human intestinal commensal. Recent evidences, however, indicate that the typical noninvasive nature of resident E. coli can be reversed under specific circumstances even in the absence of any major genomic flux. We previously engineered an E. coli strain with a mutant histone-like protein, HU, which exhibited significant changes in nucleoid organization and global transcription. Here we showed that the changes induced by the mutant HU have critical functional consequences: from a strict extracellular existence, the mutant E. coli adopts an almost obligate intracellular lifestyle. The internalized E. coli exhibits many of the prototypical characteristics of traditional intracellular bacteria, like phagosomal escape, intracellular replication, and subversion of host cell apoptosis. We suggest that E. coli K-12 can switch between widely divergent lifestyles in relation to mammalian host cells by reprogramming its cellular transcription program and without gross changes in its genomic content.

Neuroprotection conferred by astrocytes is insufficient to protect animals from succumbing to Japanese encephalitis.

Astrocytes play a key role in regulating aspects of inflammation and in the homeostatic maintenance of the central nervous system (CNS). However, the role of astrocytes in viral encephalitis mediated inflammation is not well documented. As Japanese encephalitis virus (JEV) infection is localized to neurons and considering the importance of astrocytes in supporting neuronal survival and function, we have exploited an experimental model of Japanese encephalitis (JE) to better understand the role of astrocytes in JE. Suckling mice pups were inoculated with the virus and 2 and 4 days later we analyzed a panel of molecules characteristic of reactive astrogliosis. We show that JEV infection increases the expression of astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1) and ceruloplasmin (CP). The transcript levels of growth factors produced predominantly by activated astrocytes such as nerve growth factor (NGF) and ciliary neurotrophin factor (CNTF) were elevated following JEV infection. The transcript level of brain-derived neurotrophic factor (BDNF) was also elevated following JEV infection. Both NGF and CNTF were capable of preventing ROS mediated neuronal death following in vitro JEV infection to a certain extent. Taken altogether, these data indicate that increased astrogliosis following JEV infection is accompanied by the enhanced ability of astrocytes to detoxify glutamate, inactivate free radical and produce neurotrophic factors that are involved in neuronal protection. However, this elevated physiological state of astrocyte is insufficient in conferring neuroprotection, as infected animals eventually succumb to infection. The response of astrocytes to JE can be amplified to modulate the adaptive response of brain to induce neuroprotection.

Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis.

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), IL-6, IL-1beta, TNF-alpha, and MCP-1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection.