Human isolates of Cronobacter sakazakii bind efficiently to intestinal epithelial cells in vitro to induce monolayer permeability and apoptosis.
BACKGROUND
Cronobacter sakazakii (CS) is an emerging opportunistic pathogen that causes life-threatening infections in infants. This pathogen has been implicated in the outbreaks of necrotizing enterocolitis (NEC) with associated rates of high mortality and morbidity. In this study, we compared the abilities of CS strains isolated from human and environmental sources to bind to intestinal epithelial cells and trigger apoptosis.


METHODS
CS strains were isolated from human and environmental sources and their abilities to bind to intestinal epithelial cells were determined. Monolayer permeability was determined by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) leakage. Apoptosis was examined by ApoTag and AnnexinV-7AAD staining. PKC activation was evaluated by non-radioactive PepTag assay.


RESULTS
Human isolates of CS bind to rat and human enterocytes more efficiently than environmental strains. Additionally, these strains induced increased enterocyte monolayer permeability as indicated by a decrease in TEER and an increase in transcellular leakage of exogenously added HRP. Human isolates also caused tight junction disruption and significant apoptosis of enterocytes compared with environmental strains due to increased production of inducible nitric oxide. We also observed that human CS isolates caused 2-fold increase in the activation of phosphokinase C (PKC) than environmental strains. Blocking the PKC activity in enterocytes by an inhibitor, Gö 6983, suppressed CS-mediated tight junction disruption, monolayer permeability, and apoptosis of the cells.


CONCLUSIONS
These results suggest that human isolates of CS more efficiently bind to and cause damage to intestinal epithelial cells compared with environmental strains.
Department of Gastroenterology, Children's Hospital Los Angeles, Los Angeles, California 90027, USA.
Quin
Q
Liu
Rahul
R
Mittal
Claudia N
CN
Emami
Carol
C
Iversen
Henri R
HR
Ford
Nemani V
NV
Prasadarao
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001422:Bacterial Adhesion / Q000502:physiology*; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000502:physiology; D044083:Cronobacter sakazakii / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D004756:Enterobacteriaceae Infections / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D020345:Enterocolitis, Necrotizing / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism / Q000382:microbiology*; D004781:Environmental Exposure; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000166:cytology / Q000382:microbiology; D008565:Membrane Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D014774:Virulence; D062826:Zonula Occludens-1 Protein
D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C