Salmonella enterica serovar Typhimurium-induced internalization and IL-8 expression in HeLa cells does not have a direct relationship with intracellular Ca(2+) levels

Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4467, USA.
Microbes and Infection (Impact Factor: 2.86). 06/2009; 11(10-11):850-8. DOI: 10.1016/j.micinf.2009.05.003
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The invasion-associated type III secretion system (T3SS-1) of S. Typhimurium is required to initiate and sustain an acute inflammatory response in the intestine. We investigated the relationship of S. Typhimurium T3SS-1-induced IL-8 expression and invasion with intracellular Ca(2+) mobilization in HeLa cells. Compared to the sipAsopABDE2 mutant, strains carrying a mutation in sipA, or mutations in sopABDE2 induced higher levels of IL-8 and greater bacterial internalization despite the fact that these mutants elicited similarly low intracellular concentrations of Ca(2+). Likewise, complemented sipAsopABDE2 mutant with sopE2 did not affect intracellular Ca(2+) concentrations or IL-8 expression, but significantly increased bacterial internalization. Treating HeLa cells with the calcium chelator BAPTA-AM or with D-BAPTA-AM, a derivative with greatly reduced Ca(2+) chelating activity, yielded strong evidence that BAPTA-AM does not affect invasion and inhibits IL-8 secretion by a calcium-dependent mechanism. These findings suggest that, although wild-type S. Typhimurium-induced IL-8 expression and bacterial internalization in HeLa cells coincides with increased cytosolic Ca(2+), the differing levels of IL-8 and invasion induced by strains carrying different effector proteins are unrelated to levels of intracellular Ca(2+).

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    • "Cells (from ATCC) were grown in Dulbecco's Modified Eagle Medium (DMEM) (Hyclone) supplemented with 10% (20% for Caco-2 cells) Fetal Bovine Serum (FBS) (Sigma). HeLa cells are a commonly used cell line for invasion and pedestal research [12], [13], [38], [39], [40]. We opted to use them due to their flat morphology and ease of imaging. "
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    ABSTRACT: Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.
    PLoS ONE 05/2011; 6(5):e19940. DOI:10.1371/journal.pone.0019940 · 3.23 Impact Factor
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    • "Listeria monocytogenes and Campylobacter jejuni induce intracellular Ca 2þ changes in host cells [1] [21], but while chelation of intracellular Ca 2þ interferes with C. jejuni internalization [1], it has no effect on L. monocytogenes invasion [21]. Contradictory results regarding the dependence on cytosolic Ca 2þ changes for intracellular invasion by Salmonella typhimurium and stimulation of IL-8 production have been also reported [2] [22]. "
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