Melanie A. Sherman

Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.

TLR Signaling Mediated by MyD88 Is Required for a Protective Innate Immune Response by Neutrophils to Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88−/− mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.

Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene

Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water‐borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non‐pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin‐filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif‐1 and egl‐9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek‐1, mek‐1, mev‐1, pgp‐1,3 and vhl‐1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Moreover we have found that C. elegans genes controlling lifespan (daf‐2, age‐1 and daf‐16), also mediate susceptibility to EPEC. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.

Interleukin-1 Receptor Signaling Protects Mice from Lethal Intestinal Damage Caused by the Attaching and Effacing Pathogen Citrobacter rodentium

ABSTRACT Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to the intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes a breach of the intestinal epithelial barrier, leading to colitis via a vigorous inflammatory response resulting in diarrhea and a protective antibody response that clears the pathogen. Here we show that interleukin-1 receptor (IL-1R) signaling protects mice following infection with C. rodentium. Upon infection, mice lacking the type I IL-1R exhibit increased mortality together with severe colitis characterized by intramural colonic bleeding and intestinal damage including gangrenous mucosal necrosis, phenotypes also evident in MyD88-deficient mice. However, unlike MyD88−/− mice, IL-1R−/− mice do not exhibit increased pathogen loads in the colon, delays in the recruitment of innate immune cells such as neutrophils, or defects in the capacity to replace damaged enterocytes. Further, we demonstrate that IL-1R−/− mice have an increased predisposition to intestinal damage caused by C. rodentium but not to that caused by chemical irritants, such as dextran sodium sulfate. Together, these data suggest that IL-1R signaling regulates the susceptibility of the intestinal epithelia to damage caused by C. rodentium.

Mast cells limit systemic bacterial dissemination but not colitis in response to Citrobacter rodentium.

ABSTRACT Enteropathogenic Escherichia coli and enterohemorrhagic E. coli cause an inflammatory colitis in human patients characterized by neutrophil infiltration, proinflammatory cytokine expression, and crypt hyperplasia. Citrobacter rodentium causes a similar colitis in mice and serves as a model for enteropathogenic E. coli infection in humans. C. rodentium induces systemic T-cell-dependent antibody production that facilitates clearance of the bacteria and protects the host from reinfection. The role of innate immune cells in infectious colitis, however, is less well understood. In this study, we have determined the role of mast cells in the inflammatory response and disease induced by C. rodentium. Mice deficient in mast cells exhibit more severe colonic histopathology and have a higher mortality rate following infection with C. rodentium than do wild-type animals. Despite unimpaired neutrophil recruitment and lymphocyte activation, mast cell-deficient mice have a disseminated infection evident in crucial organ systems that contributes to sepsis. Importantly, mast cells also have the capacity to directly kill C. rodentium. Together, these results suggest that mast cells protect the host from systemic infection by reducing the bacterial load and preventing dissemination of the bacterium from the colon.

Citrobacter rodentium lifA/efa1 Is Essential for Colonic Colonization and Crypt Cell Hyperplasia In Vivo

ABSTRACT Previously, we have identified a large gene (lifA, for lymphocyte inhibitory factor A) in enteropathogenic Escherichia coli (EPEC) encoding a protein termed lymphostatin that suppresses cytokine expression in vitro. This protein also functions as an adhesion factor for enterohemorrhagic E. coli (EHEC) and Shiga toxin-producing E. coli and is alternatively known as efa1 (EHEC factor for adherence 1). The lifA/efa1 gene is also present in Citrobacter rodentium, an enteric pathogen that causes a disease termed transmissible murine colonic hyperplasia (TMCH), which induces colitis and massive crypt cell proliferation, in mice. To determine if lifA/efa1 is required for C. rodentium-induced colonic pathology in vivo, three in-frame mutations were generated, disrupting the glycosyltransferase (GlM12) and protease (PrMC31) motifs and a domain in between that does not encode any known activity (EID3). In contrast to infection with wild-type C. rodentium, that with any of the lifA/efa1 mutant strains did not induce weight loss or TMCH. Enteric infection with motif mutants GlM12 and PrM31 resulted in significantly reduced colonization counts during the entire 20-day course of infection. In contrast, EID3 was indistinguishable from the wild type during the initial colonic colonization, but cleared rapidly after day 8 of the infection. The colonic epithelium of all infected mice displayed increased epithelial regeneration. However, significantly increased regeneration was observed by day 20 only in mice infected with the wild-type in comparison to those infected with lifA/efa1 mutant EID3. In summary, lifA/efa1 is a critical gene outside the locus for enterocyte effacement that regulates bacterial colonization, crypt cell proliferation, and epithelial cell regeneration.

Expression of UDP-glucuronosyltransferase isoform mRNAs during inflammation and infection in mouse liver and kidney.

Inflammation or infection down-regulates the activity and expression of cytochrome P450 (P450) enzymes involved in hepatic drug clearance, possibly altering drug effectiveness and leading to toxicity. The regulation of UDP-glucuronosyltransferases (UGTs) in inflammation and infection is less well characterized. To determine the response of hepatic and renal UGTs during inflammation and infection, mice were administered either saline or 1 mg/kg lipopolysaccharide (LPS) (16 h), or Citrobacter rodentium by oral gavage (6 days). Hepatic mRNA expression of UGT1A1, 1A9, and 2B5 was similarly down-regulated after LPS exposure and C. rodentium infection, whereas UGT1A2 and 1A6 mRNAs were unchanged. Effects of C. rodentium infection did not require a functional Toll-like receptor 4. Conversely, renal UGT isoforms were relatively unaffected, except for UGT2B5 induction after LPS treatment. Regulation of UGTs during the inflammatory response exhibits similarities to and differences from regulation of P450s, and may be cytokine-mediated.

STAT6-independent production of IL-4 by mast cells.

The acquisition of an IL‐4‐producing phenotype in Th2 cells requires IL‐4 signaling through the STAT6 pathway during T cell differentiation. In this study we demonstrate that, unlike in naive T cells, IL‐4 is not necessary for the development of an IL‐4‐producing phenotype in mast cells. Bone marrow‐derived mast cell precursors from STAT6–/– mice can differentiate into mature cells that express IL‐4 levels comparable to those of wild‐type mast cells. In differentiated mast cells, activation in the presence of neutralizing anti‐IL‐4 antibodies or mutation of the consensus STAT6 sites does not diminish IL‐4 promoter activity, indicating thatIL‐4 is not required for active transcription. Taken together, these data suggest that mast cell IL‐4 production is not STAT6 dependent, providing evidence that these cells could generate IL‐4 needed for the initiation and amplification of an effective Th2 immune response.

HEPATIC AND RENAL CYTOCHROME P450 GENE REGULATION DURING CITROBACTER RODENTIUM INFECTION IN WILD-TYPE AND TOLL-LIKE RECEPTOR 4 MUTANT MICE

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor α (TNFα) mRNAs were significantly increased in infected HeOu mice, whereas only TNFα mRNA was significantly increased in HeJ mice. Hepatic α1-acid glycoprotein was induced in both groups, whereas α-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

IL-4 Induces the Proteolytic Processing of Mast Cell STAT6

IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells.