|
The epithelial cells that line the intestinal tract
are occasionally exposed to enteric pathogens, such as Salmonella.
Epithelial cells can recognize foreign bacterial products and may
respond by prompting local inflammation and programmed cell death
(apoptosis). Previous research on Salmonella-gut interactions has
primarily focused on the inflammatory response. The protein flagellin
is known to elicit pro-inflammatory and cell survival gene expression,
and aflagellate Salmonella do not induce such responses. This study
investigates the activation of apoptotic pathway intermediates-caspases-in
response to infection by wild type Salmonella and an aflagellate
mutant. We modeled Salmonella-gut interactions in vitro by infecting
human HeLa cells with wild type Salmonella strain SL3201 or an aflagellated
mutant strain. Using Western blot analysis and fluorescent active-caspase
detection reagents, we observed that caspase-8, caspase-9, and caspase-3
were activated by both strains. However, infection with the mutant
bacteria yielded an increased and more stable level of activation.
We attribute the transient nature of caspase activation by wild
type Salmonella to the pro-inflammatory, anti-apoptotic response
stimulated by flagellin. These results suggest that infection with
non-flagellated pathogens causes more apoptosis than infection with
flagellated types. This also implies that during natural Salmonella
infection, an inflammatory response attenuates epithelial tissue
damage and allows resolution of the infection.
Membrane-embedded protein receptors within the epithelium
recognize characteristic bacterial products, termed 'pathogen-associated
molecular patterns' (PAMPs). During infection, PAMPs and cellular
stress stimulate the cell to activate intracellular signaling pathways.
These include the proinflammatory NF-B pathway, which is coupled
with pathways that lead to programmed cell death-apoptosis. Caspases
are proteolytic intermediates and effectors in the apoptotic pathway.
They remain inactive as procaspases until they are cleaved by other
proteases or each other, following the recognition of a pro-apoptotic
signal. Initiator caspases relay and magnify the signal by cleaving
downstream effector caspases, which in turn trigger the physiologic
changes of cell death [1]. Salmonella enterica serovar typhimurium
is a Gram-negative, intracellular pathogen, commonly associated
with food-borne enteritis, contributing to 800,000 to 4 million
infections in the US every year [2]. These bacteria are known to
elicit an inflammatory response via the protein flagellin and the
NF-B pathway [3]. They also initiate apoptosis, but the mechanisms
of this response are not well understood.
Experimental Co-culture
Propagating cultures of human cervical carcinoma (HeLa) cells were
maintained in DMEM-based culture media. Cells were grown to confluency
on 6-well plastic plates or on glass coverslips. For bacterial infections,
wild type and fliC/fljB mutant type S. typhimurium were added at
a multiplicity of infection (MOI) of 10-30 organisms per cell. Controls
were treated with Hanks' Balanced Salts Solution (HBSS) with and
without TNF& (10 ng/mL). Cells were lysed in SDS Laemmli buffer.
Detection of Caspase Activity
Rates and intensities of caspase activation were analyzed semi-quantitatively,
using (1) Western blotting with anti-active- caspase antibodies,
and (2) carboxyfluorescein (FAM) labeled caspase inhibitors (APO
LOGIXT, Cell Technology). The APO LOGIXT inhibitors are inherently
fluorescent and bind to the active forms of specific caspases. APO
LOGIXT reagents were added for the final hour of infection, and
cells were washed, then observed by confocal fluorescence microscopy
at 600 nm.
Cells exposed to the aflagellate (CB) strain exhibited
a consistent rise in caspase activation, while cells treated with
the wild type demonstrated markedly attenuated activation. Of the
three caspases observed, caspase-8 was activated more rapidly than
the other two. Detection of cleaved caspase-8 shows an increase
in activation after one hour of infection with both strains. Activation
remained strong in CB treatments, but weakened after 6 hours with
wild type. Compared to the baseline (Hanks'), little overall activation
of caspase-9 was observed. A slight peak of activation is shown
at 3 hours in both treatments. The decrease in band strength at
6 and 9 hours of CB may be due, in part, to gel loading artifact.
Active cleaved caspase-3 was observed at 3 hours. Activation subsided
in wild type treatments. Due to potential loading artifact, it is
not possible to determine the extent of the decrease in CB treatments.
Compared to wild type Salmonella, an isogenic aflagellated strain
more effectively activates and maintains the caspase cascade in
human HeLa cells. Treatment with aflagellated Salmonella causes
activation of caspase-8 more rapidly and extensively than caspase-9,
suggesting that the mutant bacteria utilize the extrinsic apoptotic
pathway, even though they lack flagella. Infection with wild type
Salmonella leads to reduced caspase activation. This is likely due
to the potent stimulation of the NF-B pathway by flagellin, and
the resulting upregulation of pro-inflammatory, anti-apoptotic effector
proteins. This study suggests that epithelial cells respond to wild
type Salmonella by prompting an immuno-inflammatory response, thereby
limiting cell death and tissue damage while still eliminating the
source of infection. Further research may demonstrate that certain
species of non-flagellated pathogens cause more apoptosis-therefore
more tissue damage-than do flagellated species or strains.
This research was funded by Howard Hughes Medical Institute Grant
No. 52003727. Many thanks to the Summer Undergraduate Research at
Emory (SURE) program for making the opportunity possible, and to
Brigid Batten for answering countless questions about Western blots!
References
1. Alberts B, et al. Molecular Biology of the Cell, 4th edn. New
York: Garland; 2002.
2. http://www.cdc.gov/od/oc/media/fact/salmonella.htm
3. Zeng H, Carlson AQ, Guo Y, et al. J Immun 2003; 171:3668-367
4. Salmonella picture found at http://www.innovations-report.com/bilder_neu/5573_salm.jpg
Have you heard of Salmonella? You probably know that you shouldn't
eat raw eggs or poultry, since you run the risk of food poisoning.
Well, this summer I studied the details about how Salmonella bacteria
make people sick to their stomachs. I grew human cells on petri
dishes, then poured on billions of bacterial cells, and observed
the cells' reactions. I was particularly watching for patterns in
the cells that committed suicide once they realized what was going
on. Proteins on the surface of human gut cells recognize the bacteria,
and use other proteins--caspases--to send a message to the rest
of the cell, and to make the cell die. These caspases were the target
of my research. I learned that fewer cells commit suicide when exposed
to Salmonella with tails (flagella) then when exposed to Salmonella
without tails. Since Salmonella with tails are more common, this
shows that the human body has learned how to handle Salmonella without
allowing many cells to die.
Western blot, Cell tissue, culture, bacterial infection, fluorescent
microscopy.
|
