A Salmonella enterica serovar Typhi plasmid induces
rapid and massive apoptosis in infected macrophages
Shuyan Wu, Yuanyuan Li, Yang Xu, Qiong Li, Yuanyuan Chu, Rui Huang and Zhenghong Qin
pRST98is a chimeric plasmid isolated from Salmonella enterica serovar Typhi (S. typhi) that mediates the functions of drug resistance
and virulence. Previously, we reported that Salmonella plasmid virulence (spv) genes were present in S. typhi. In our current study, we
investigated whether plasmid pRST98exhibits significant cytotoxicity in macrophages. pRST98was transferred into the avirulent
Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create the transconjugant pRST98/RIA. The standard S.
Results showed that macrophages infected with strain pRST98/RIA displayed greater levels of apoptosis than those infected with RIA
and that pRST98may increase bacterial survival in macrophages. Further studies showed that the pRST98-induced death of
macrophages was associated with the loss of mitochondrial membrane potential and that pRST98may activate caspase-9 and then
caspase-3. The research data indicate that the virulence of bacteria that contain the pRST98plasmid is enhanced; the presence
of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage
Cellular & Molecular Immunology (2010) 7, 271–278; doi:10.1038/cmi.2010.17; published online 17 May 2010
Keywords: apoptosis; macrophage; plasmid; Salmonella enterica serovar Typhi
Typhoid fever is a classic systemic infection caused by Salmonella
problem in developing countries. Recent dataestimate that22million
(range 16–33 million) cases occur each year, resulting in 216 000
deaths, predominantly in school-age children and young adults.1In
addition, strains of S. typhi have become resistant to chloramphenicol
and other recommended antibiotics (ampicillin, cotrimoxazole and
even ciprofloxacin). These resistant strains have become prevalent in
several areas of the world. Polluted water is the most common source
of typhoid transmission. The highest incidence of typhoid usually
occurs where large populations are exposed to contaminated water
supplies. In China, the morbidity of typhoid fever has been under
control since 1990, but there are still localized areas where the disease
incidence remains high and there are occasional outbreaks.
A pandemic of multidrug resistant S. typhi occurred in China in the
mid to late 1980s, with 13 provinces and cities affected. In Suzhou,
591 strains of S. typhi were isolated from the blood of patients; these
strains were examined for antimicrobial susceptibility by our lab.More
than 80% of the isolates were multidrug resistant. This drug resistance
was caused by a large, conjugative plasmid of 98.6 MDa (150 kb) that
is classified in incompatibility group C.2The S. typhi plasmid was
designated pRST98and is known to mediate bacterial multidrug resist-
ance to chloramphenicol, streptomycin, trimethoprim, sulfonamide,
gentamycin, neomycin, kanamycin, cephalosporin, ampicillin, carbe-
nicillinandtetracycline.Patients infectedwithS.typhithat waspositive
for pRST98had severe symptoms and exhibited complications with
high mortality rates. However, only one plasmid existed in all of these
isolates. This led us to presume that pRST98may be a mosaic-like
plasmid that is responsible for drug resistance and increased virulence
been the subject of much research in recent years. Although the exact
contribution of virulence plasmids to pathogenesis is still a matter of
debate, progress has been made in identifying regions of the plasmid
that are necessary for conferring virulence. Kurita et al.3identified a
highly conserved region of 8 kb, designated the Salmonella plasmid
virulence (spv) genes; these genes are present in the plasmids of all
passes five genes, the first gene, spvR, encodes a positive activator for
the following four genes, spvABCD. The spv genes appear to promote
the rapid growth and survival of Salmonella within the host cells that
mental animals.4In 2005, we identified the virulence genes on pRST98
by Southern blot and DNA sequence analyses, which demonstrated
also present on pRST98, a plasmid carrying the genes encoding the
properties of drug resistance and virulence in S. typhi.5
Medical College of Soochow University, Suzhou, China
Correspondence: Dr R Huang and Dr ZH Qin, Medical College of Soochow University, No. 199, Ren Ai Road, Suzhou 215123, China.
Received 6 December 2009; revised 4 February 2010; accepted 22 February 2010
Cellular & Molecular Immunology (2010) 7, 271–278
? 2010 CSI and USTC. All rights reserved 1672-7681/10 $32.00
relationship between autophagy and apoptosis are now being carried
out in our lab.
In summary, characterization of the chimeric plasmid pRST98iso-
lated from S. typhi is potentially important for understanding the
virulence mechanism of this pathogen. Our study indicated that the
potential of cytotoxicity mediated through the mitochondrial path-
wayisprimarily involvedinplasmid pRST98-induced apoptosis. These
are the first results to show that the plasmid isolated from S. typhi can
enhance the virulence of host bacteria by mediating the apoptosis of
the macrophage host cells; this study provides important information
on the mechanisms of S. typhi-induced macrophage apoptosis.
We are grateful to Professor Roy Curtiss III, The School of Life Sciences,
11 and RIA. We also thank Professor Jie Yan, Zhejiang University, China, for
providingmurine macrophage-like cell line J774A.1.This study was supported
by the Natural Science Foundation of China (No. 30972768), the Special and
General Postdoctoral Science Foundation of China (No. 200902529 and
No. 20080430178), the Natural Science Foundation of Jiangsu High Education
Institute of China (No. 08KJB310009) and the Social Development Science
Foundation of Suzhou City of China (No. SS08025).
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Figure6 Caspase-9 andcaspase-3 activitiesin culturesupernatantsofJ774A.1
Values are expressed as means6standard deviation of seven different observa-
tions. *Significantly different (P,0.01)from uninfected control andRIA-infected
cells. S. typhimurium, Salmonella enterica serovar Typhimurium.
Figure 7 Bacterial survival in J774A.1 cells. Bacterial Log CFU per 105macro-
phage cells (yaxis) and time afteraddition ofamikacin (x axis) are indicated. The
results are presented as the mean6standard error of the mean. CFU, colony-
S. typhi plasmid induces macrophage apoptosis
Wu et al
Cellular & Molecular Immunology