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*
The Inflammation Program and Department of Medicine, University of Iowa and the Veterans’ Affairs Medical Center, Iowa City, IA 52242; and
Department of Microbiology, University of Iowa College of Medicine, Iowa City, IA, 52242
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific virulence factors encoded within Salmonella pathogenicity islands (SPI)4 enable the bacteria to evade or overcome antimicrobial defenses at various stages of infection. The expression of these virulence determinants is precisely regulated both temporally and spatially (reviewed in Ref. 4): SPI-1 genes are transcribed before bacteria breach the intestinal epithelium (5), and SPI-2 products are required at later stages of infection for the bacteria to reach and survive within the lymphatics and bloodstream. During this second stage of infection, S. typhimurium seems to elude the host immune response by residing within macrophages.
SPI-1 and SPI-2 each encode a type III secretion system (TTSS) that translocates bacterial proteins into target eukaryotic cells (6). Once inside the host cell, bacterial proteins can modify or promote cellular events and thus interfere with host cell biological responses. SPI-1-encoded secreted proteins induce membrane ruffling of epithelial cells, thereby promoting bacteria uptake via a macropinocytic process (7, 8). Although SPI-2 is associated with survival within macrophages and subsequent systemic infection (2, 9), much less is known about the mechanisms by which SPI-2-encoded proteins alter host responses to Salmonella. However, the decreased survival of SPI-2 mutants in macrophages (10, 11, 12, 13) suggests that SPI-2-encoded secreted proteins compromise the bactericidal function of macrophages.
Phagocytes use both O2-dependent and O2-independent mechanisms to kill ingested bacteria, and many successful pathogens have developed multiple strategies to avoid or withstand exposure to toxic agents present in the phagosome. Recently published work indicates that S. typhimurium strains deficient in components of the SPI-2-encoded TTSS are less virulent in wild-type (WT) mice but not in mice that lack functional NADPH oxidase activity, suggesting that S. typhimurium evades or withstands the phagocyte NADPH oxidase in an SPI-2-dependent manner (13).
To determine the mechanisms by which oxidant production is subverted during phagocytosis of S. typhimurium by human macrophages, we analyzed the subcellular distribution of both the membrane component, flavocytochrome b558 (cyt b558), and the cytosolic components, p47phox and p67phox, of the NADPH oxidase during infection of monocyte-derived macrophages (MDMs) by WT or several SPI-2 mutant strains of S. typhimurium. We found that most phagosomes containing S. typhimurium that express a complete SPI-2 TTSS lacked cyt b558, thereby thwarting successful assembly of the NADPH oxidase and avoiding O2-derived antimicrobial agents. We discuss how preventing NADPH oxidase assembly may contribute to the ability of S. typhimurium to circumvent successfully host microbicidal pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dulbecco’s PBS (DPBS), RPMI 1640, MEM
,
L-glutamine, FBS, and goat serum were purchased from Life
Technologies (Grand Island, NY). Lab-Tek 8 well-chamber slides were
obtained from Nalge Nunc International (Naperville, IL). A mixture of
three murine mAbs to cyt b558 was used
in immunofluorescence microscopy. 7D5, a generous gift from Michio
Nakamura (Institute of Tropical Medicine, Nagasaki, Japan), recognizes
an extracellular conformational epitope on
gp91phox expressed only by the assembled
heterodimer (14). The Abs recognizing
gp91phox (54.1) and
p22phox (44.1) were the generous gifts from A.
Jesaitis, M. Quinn, and J. Burritt (Montana State University,
Bozeman, MT) and recognize linear epitopes on the cytosolic domain of
the respective subunits of cyt b558
(15). Anti-p47phox and
p67phox polyclonal Abs were previously described
(16). Texas Red-coupled anti-mouse and anti-rabbit
secondary Abs were obtained from Molecular Probes (Eugene, OR), and
Cy5-conjugated anti-mouse secondary Ab from Jackson ImmunoResearch
(West Grove, PA). Kodak Ektachrome 400 ASA color slide film was
purchased from Eastman Kodak (Rochester, NY). All other reagents were
obtained from Sigma (St. Louis. MO).
Bacterial strains and growth conditions
Chromosomal mutations were introduced in SL1344 S. typhimurium in genes coding for the TTSS apparatus (secretion system apparatus or ssa) or in putative effector proteins (secretion system effector or sse). The S. typhimurium ssaT mutant used in this study was made using S. typhimurium strain P9B7 ssaT::mTn5 obtained from David Holden (17). The ssaT::mTn5 mutation was moved by P22-mediated transduction to S. typhimurium strain SL1344. The desired transductant was designated S. typhimurium JK14. The S. typhimurium sseD mutant was constructed using the following method. The sseD gene was amplified from the SL1344 chromosome using primers sseD1 (5'-GAAACGGCAATGATGTGCGG-3') andsseD2 (5'-CTGCCATGAGGCGTAACCAC-3'). The 2-kb DNA product was cloned into pGEM-T Easy (Promega, Madison, WI) and a blunt-ended aphT gene encoding kanamycin resistance was ligated into the middle of the sseD gene digested with EcoRV. The sseD-aphT construct was cloned into the pBDJ129 vector, followed by selection for allelic exchange of the mutated sseD into the SL1344 chromosome (18, 19). This strain was designated S. typhimurium JK22. SseD protein was not expressed from JK22, as determined by Western blot analysis. In addition, the mutation resulted in severe attenuation of mouse virulence (19). JK22 was complemented by a plasmid carrying the appropriate gene, indicating that the mutation did not have polar effects on downstream genes (19). Plasmid pGREEN3 (CamR) constitutively expressing green fluorescent protein (GFP) was introduced into SL1344 and JK22 to visualize bacteria in the infection experiments.
Bacteria were grown overnight from a single colony in minimum magnesium medium (pH 5.5) to an approximate A600 of 0.5. These growth conditions induce the expression of SPI-2 TTSS (19).
Preparation of macrophages monolayers
MDMs. PBMC were isolated from human venous blood obtained from consenting adults in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. Mononuclear cells were cultured in Teflon wells for 5–7 days at a density of 2 x 106 cells/ml in RPMI 1640 complemented with 20% fresh autologous serum, as previously described (20). Cells were then washed three times in RPMI 1640 and resuspended at 5 x 106 cells/ml in RPMI 1640 supplemented with 10 mM HEPES and 10% autologous serum. Macrophages were purified by adherence to eight-well chamber slides using 2 x 106 mononuclear cells/well. After 2 h of incubation at 37°C in 5% CO2, nonadherent cells were removed by washing the monolayer three to four times in RPMI 1640 before infecting the cells with S. typhimurium.
Mouse peritoneal macrophages.
Resident macrophages were harvested by peritoneal lavage from female
CD-1 mice and plated in MEM supplemented with 10% heat inactivated
FBS, 1% L-glutamine, and 100 U/ml penicillin G and 100
µg/ml streptomycin. After 2 h at 37°C lymphocytes were removed
by washing and adherent macrophages were incubated overnight at 37°C
in antibiotic-free medium before use.
Intraphagosomal superoxide generation
MDMs were plated on chamber slides and peritoneal
macrophages were plated on glass coverslips to achieve 
50%
confluence. S. typhimurium SL1344, sseD,
ssaT strains, or zymosan particles were washed, dispersed in
HEPES-RPMI 1640 containing 1 mg/ml nitroblue tetrazolium (NBT), and
then added to macrophages to achieve a ratio of 3 zymosan/cell or 25
bacteria/cell. Phagocytosis was synchronized using centrifugation
(600 x g, 3 min, 16°C) followed by a 60-min
incubation at 37°C. Samples were washed three times with DPBS,
counterstained with Wright-Giemsa (SureStain; Fisher, Pittsburgh, PA),
and then sealed using Permount (Fisher). Light microscopy was performed
using a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) and
samples were photographed using ASA 400 color slide film. Phagosomes
were monitored for the presence of blue-black formazan precipitate
formed by the reduction of NBT by superoxide anion. At least 300
phagosomes were scored per experiment in triplicate samples.
Synchronized phagocytosis
GFP-expressing S. typhimurium SL1344, sseD, or ssaT strains were suspended in complete medium (RPMI 1640 with 10 mM HEPES and 10% heat-inactivated FBS) at a concentration of 2.5 x 106 bacteria/ml. Phagocytosis was synchronized by centrifugation of bacteria (106 bacteria/well) onto the macrophage monolayer (2 min, 600 x g, 12°C), followed by incubation at 37°C in 5% CO2 for the desired amount of time. Cells were then stained and/or processed for immunofluorescence microscopy (see below).
Assessment of bacterial growth
Replication of S. typhimurium inside macrophages was evaluated by immunofluorescence and confocal microscopy. This approach was recently described by Hensel and collaborators in the back cover of a recent issue of Molecular Microbiology (Vol. 36, issue 5, 2000). Following centrifugation of S. typhimurium onto the macrophage monolayer, chamber slides were incubated at 37°C in 5% CO2 for 30 min. Gentamicin was then added at a final concentration of 6 µg/ml and incubation was extended for 90 min. We verified that this treatment effectively killed noningested bacteria by quantitating CFUs after exposing bacteria to gentamicin (5–100 µg/ml) for 10–60 min (data not shown). Furthermore, 85–95% of cell-associated WT or mutant S. typhimurium was intracellular after 5 min of phagocytosis at 37°C, as judged by confocal microscopy.
Following the 90-min treatment with gentamicin, cells were washed once in RPMI 1640 and processed for microscopy (2-h time point) or further incubated at 37°C in 5% CO2 in gentamicin-free complete medium for 4 or 8 h (6- or 10-h time points, respectively) to minimize antibiotic-mediated effects on intracellular S. typhimurium. Cells were then stained with Wright-Giemsa and examined using a Zeiss Axioplan2 photomicroscope or processed for immunofluorescence microscopy. Bacteria were quantified from 100 infected macrophages for each strain in each of three to six independent experiments. The numbers obtained were compared using paired Student’s t test with significance set at p < 0.05.
Immunofluorescence microscopy and image processing
S. typhimurium-infected MDMs were fixed in 10% neutral-buffered Formalin for 15 min at 25°C and then permeabilized in 50% methanol/50% acetone for 5 min at 4°C. Fixed and permeabilized cells were rinsed in DPBS supplemented with 0.5 g/L sodium azide and 5 g/L BSA (PAB), and then blocked in PAB containing 10% heat-inactivated normal goat serum (blocking buffer) for 1 h at 25°C or overnight at 4°C. Following incubation with primary Abs (anti-p47phox or p67phox rabbit Ab and/or anti-cyt b558 mouse Ab, diluted in blocking buffer) for 1 h at 25°C, cells were washed five times in PAB, and incubated with appropriate secondary Abs (anti-mouse Cy5 and/or anti-rabbit Texas Red, diluted in blocking buffer) or rhodamine-phalloidin for an additional hour. After five washes in PAB, the slide was detached from the chamber and mounted using the Molecular Probes SlowFade Light Antifade kit (Eugene, OR). Cells were viewed using a Zeiss LSM 510 confocal microscope. Phagosomes were monitored for the presence of cyt b558 and either p47phox or p67phox. At least 30 phagosomes were scored per sample from three separate experiments. The numbers of positive phagosomes obtained at each time point were compared using paired Student’s t test with significance set at p < 0.05.
Analysis of infected MDMs lysates by immunoblotting
Noninfected and WT or sseD S. typhimurium-infected MDMs (2-h time point) were lysed by a 30-min incubation in lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 10 mM EDTA, 100 µg/ml leupeptin, 100 µg/ml pepstatin, and 1 mM PMSF), followed by solubilization in SDS sample buffer. Samples were resolved by 9% SDS-PAGE and then transferred to nitrocellulose. Membranes were blocked with 3% BSA in PBS containing 0.05% sodium azide and 0.1% Nonidet P-40 and probed with Abs against gp91phox (54.1) or p22phox (44.1). HRP-conjugated goat anti-mouse (Bio-Rad, Hercules, CA) Abs were used to detect immunoreactive proteins by ECL. The total protein content of the cellular lysate was visualized by staining with Ponceau S (Boehringer Mannheim; Ingelheim, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the SPI-2-encoded TTSS is required for survival
or growth of S. typhimurium inside phagocytes
(10, 11, 12, 13), we assessed the ability of strains of S.
typhimurium with mutations in genes encoding components of the
TTSS to replicate inside MDMs. We compared the number of WT and
sseD GFP-S. typhimurium that associated with MDMs
after 2, 6, or 10 h. Although similar numbers of WT and sseD
S. typhimurium were seen within MDMs at early time points (Fig. 1
, upper panels), after
6–10 h MDMs infected with S. typhimurium WT contained
clusters of bacteria in close proximity. These clusters of bacteria
likely reflected intracellular replication, since no clumping of
bacteria was observed at earlier time points (Fig. 1, upper
panels). In both cases, intracellular bacteria were identified
within spacious phagosomes (Fig. 1, lower panels), as
previously described (21). Confocal microscopic
examination of serial sections throughout the whole cell demonstrated
that the clusters of bacteria were intracellular (Fig. 2). Comparable numbers of bacteria were
cell associated at the 2-h time point in WT and
sseD-infected MDMs (Fig. 3A), indicating that both
strains were phagocytosed with equal efficiency. In contrast, by
10 h, sseD showed a 50% reduction of cell-associated
bacteria relative to the WT (Fig. 3B). We noted significant
heterogeneity with either WT or mutant S. typhimurium with
respect to the number of bacteria invading a given cell. However, taken
as a population, the number of intracellular bacteria was consistently
reduced in sseD-infected MDMs relative to WT-infected MDMs
(Fig. 3), suggesting that the sseD strain had a decreased
ability to replicate within macrophages when compared with the WT
strain.
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The ultrastructural data suggest that intraphagosomal accumulation
of reactive oxygen species (ROS) is reduced in phagosomes containing WT
S. typhimurium relative to phagosomes containing the SPI-2
mutant sseB (13). We found that both murine
peritoneal macrophages and human MDMs readily ingested WT S.
typhimurium and the isogenic SPI-2 mutants (sseD and
ssaT). All organisms were found in spacious phagosomes;
however, these structures tended to be smaller in MDMs than in murine
macrophages (Fig. 4). Using an
intraphagosomal NBT assay to detect production of superoxide, we found
that formazan precipitates, occurring as a result of a localized
reduction of NBT by superoxide anion, were generated inside only
13–25% of phagosomes containing WT S. typhimurium (Fig. 4). In contrast, 75–85% of phagosomes containing the SPI-2 mutants
sseD and ssaT, or control phagosomes containing
zymosan particles, were NBT positive (Fig. 4 and data not shown).
Because similar data were obtained using human and murine macrophages
and because all organisms induced formation of spacious phagosomes, the
absence of detectable superoxide in the majority of phagosomes
containing WT S. typhimurium cannot be explained by
differences in phagosome morphology or macrophage species. These
results suggest that WT S. typhimurium interfered with
superoxide generation in macrophage phagosomes in an SPI2-dependent
manner and that the functions of the SseD and SsaT proteins were
required for this process.
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The failure to detect phagosomal superoxide anion could reflect
inhibition of NADPH oxidase assembly or activity and/or rapid quenching
of ROS by WT S. typhimurium. To distinguish among these
possible mechanisms, we used immunofluorescence and confocal microscopy
to determine the distribution of the NADPH oxidase subunits in
macrophages infected with WT, ssaT, or sseD
strains (
Figs. 5–7). Our previous
studies demonstrated that the membrane component of the NADPH oxidase,
cyt b558, localizes mainly in the
plasma membrane and discrete intracellular compartments of resting
neutrophils and is enriched at the phagosomal membrane during ingestion
of zymosan or live Neisseria meningitis (22).
However, we found that cyt b558 was
absent from, or minimally present in, the phagosomal membrane of MDMs
infected with WT S. typhimurium (Figs. 5A and
6A). In contrast,
sseD and ssaT S. typhimurium-containing
phagosomes were frequently enriched for cyt
b558 (Figs. 5B,
6B, and 7A).
Seventy to 80% of the phagosomes containing the SPI-2 mutants were
enriched for cyt b558 after 1 min of
phagocytosis (Fig. 7A). At identical time points, only 40%
of phagosomes in MDMs containing WT S. typhimurium were
positive for cyt b558 (Fig. 7A). By 20 min, only 25% of WT S.
typhimurium-containing phagosomes were positive for cyt
b558 vs 80% for ssaT and
sseD strains (Fig. 7A). In addition, the
cytosolic oxidase proteins p47phox and
p67phox colocalized with cyt
b558 at the phagosomal membrane of
ssaT and sseD S. typhimurium-containing
phagosomes (Fig. 7 and data not shown), demonstrating assembly of a
functional NADPH oxidase complex. In contrast, the cyt
b558-negative WT S.
typhimurium phagosomes were not enriched for
p47phox (data not shown), confirming the
requirement for the cyt b558 as a
docking site for stable association of the cytosolic oxidase components
with the membrane (16). Immunoblots of lysates of human
MDMs infected with WT or sseD S. typhimurium were
probed to determine whether differential proteolysis of cyt
b558 might account for its absence
from phagosomes containing WT bacteria. There was neither a difference
in the amounts of full-length gp91phox or
p22phox (data not shown) nor the presence of
proteolytic fragments in MDMs infected with either strain of S.
typhimurium (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reduced phagosomal detection of ROS in phagocytes containing WT S. typhimurium might be explained by the action of SPI-2 at different levels of the phagocyte biological response. Decreased detection of ROS could reflect scavenging by bacterial copper, zinc-superoxide dismutase, or inhibition of the activity of the assembled oxidase through disturbance of electron transfer or proteolysis of oxidase subunits. Finally, bacterial factors may interfere earlier in the course of ROS production to prevent assembly of the NADPH oxidase complex by inhibiting the recruitment of the oxidase subunits to the phagosome. Using synchronized phagocytosis and confocal immunofluorescence microscopy to localize oxidase components in S. typhimurium WT or SPI-2 mutant-infected macrophages, we found that S. typhimurium acts very early during the assembly of the NADPH oxidase on the nascent phagosome to prevent ROS production in the phagosome lumen. Our data indicate that the absence of NADPH oxidase activity from S. typhimurium-containing phagosomes reflected the lack of enrichment of the cyt b558 in the phagosomal membrane and that this effect required a functional SPI-2-encoded TTSS.
In addition to generation of ROS, macrophages employ several other mechanisms to kill ingested bacteria, including acidification of the phagosome via acquisition of vacuolar H+ ATPase, and delivery of cytolytic enzymes to the phagosome lumen via fusion with intracellular vesicles of the endosomal or lysosomal pathway. Several pathogenic microorganisms, including Salmonella, can interfere with the fusion between phagosomes and lysosomes, thereby modulating the phagosomal compartment in which they reside (25, 26). An SPI-2-secreted protein, SpiC, is transferred into the host cytosol and contributes to the ability of S. typhimurium to interfere with intracellular membrane trafficking (27). Our results support the notion that the assembly of a functional NADPH oxidase is an early and critical element in recruiting bactericidal agents to the organism-containing vacuole. The efficient assembly of phagosomal NADPH oxidase in MDMs infected by S. typhimurium sseD strain, but not WT S. typhimurium, was associated with a reduction in bacterial replication that was detected as soon as 6 h after infection. The ability of WT S. typhimurium to interfere with the early O2-dependent antimicrobial response of MDMs may represent a mechanism by which Salmonella ensures its survival in the early phagosome.
The SPI-2-dependent exclusion of NADPH oxidase subunits from the phagosome may have implications beyond inhibition of ROS generation. It has been known for some time that acidification of the S. typhimurium phagosome is delayed and attenuated. Interestingly, several investigators have shown that cyt b558 may translocate H+ or may signal activation of a proton-conducting entity (28, 29, 30, 31, 32, 33). Therefore, it is tempting to speculate that exclusion of cyt b558 from the phagosomal membrane may be responsible, at least in part, for the attenuated acidification of the S. typhimurium phagosome. It is likely that the overall fate of S. typhimurium in the macrophage reflects both early steps coinciding with phagosome formation as well as later events associated with phagosome maturation.
The precise mechanisms by which the NADPH oxidase normally assembles at the plasma membrane of phagocytosing macrophages have not been fully elucidated. In contrast to polymorphonuclear leukocytes, in which cyt b558 is present not only at the plasma membrane but also in the membrane of specific granules, macrophages express the vast majority of cyt b558 at the plasma membrane (34). Our finding that WT- and SPI-2 mutant-containing phagosomes were differently enriched for cyt b558 and p47phox suggests that S. typhimurium interfered with the signaling events that mediate organization and assembly of the NADPH oxidase. We hypothesize that cyt b558 enrichment at the phagosomal membrane of macrophages occurs via early clustering or lateral diffusion of the transmembrane cyt b558 toward the site of initial engagement of the bacteria at the surface of the phagocyte. The effectors of SPI-2 TTSS may disrupt the proximal signaling that normally targets cyt b558 to specific regions of the membrane engaged in phagosome formation. Although our data do not directly test this hypothesis, there is substantial evidence demonstrating dramatic reorganization of the phagocyte plasma membrane during both phagocytosis and chemotaxis (35, 36, 37). Remodeling of the surface topography need not be limited to these agonist-dependent events and may well be critical for precise spatial control of ROS generation. Alternatively, components of the TTSS may actively exclude cyt b558 from the phagosomal membrane early in host cell invasion, rapidly degrade, and/or modify cyt b558, the net result being the loss of a functional NADPH oxidase complex at the phagosomal membrane. Further characterization of the strategies successfully used by pathogens to foil cellular antimicrobial defenses will provide important insights into mechanisms underlying normal phagocyte responses.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology, University of Minnesota, 1460 Mayo Building, Box 196, 420 Delaware Street SE, Minneapolis, MN 55455-0312.
3 Address correspondence and reprint requests to Dr. William M. Nauseef, Department of Internal Medicine, University of Iowa, 200 Hawkins Drive, SW54 GH, Iowa City, IA 52242.
4 Abbreviations used in this paper: SPI, Salmonella pathogenicity island; TTSS, type III secretion system; cyt b558, flavocytochrome b558; DPBS, Dulbecco’s PBS; GFP, green fluorescent protein; MDM, monocyte-derived macrophage; NBT, nitroblue tetrazolium; ROS, reactive oxygen species; ssa, secretion system apparatus; sse, secretion system potential effector; WT, wild type; PAB, PBS containing 0,5 g/L sodium azide and 5 g/L BSA.
Received for publication October 20, 2000. Accepted for publication February 28, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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K.-i. Uchiya and T. Nikai Salmonella enterica Serovar Typhimurium Infection Induces Cyclooxygenase 2 Expression in Macrophages: Involvement of Salmonella Pathogenicity Island 2 Infect. Immun., December 1, 2004; 72(12): 6860 - 6869. [Abstract] [Full Text] [PDF]
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J. A. Tobar, P. A. Gonzalez, and A. M. Kalergis Salmonella Escape from Antigen Presentation Can Be Overcome by Targeting Bacteria to Fc{gamma} Receptors on Dendritic Cells J. Immunol., September 15, 2004; 173(6): 4058 - 4065. [Abstract] [Full Text] [PDF]
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J. W. IJdo and A. C. Mueller Neutrophil NADPH Oxidase Is Reduced at the Anaplasma phagocytophilum Phagosome Infect. Immun., September 1, 2004; 72(9): 5392 - 5401. [Abstract] [Full Text] [PDF]
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D. Drecktrah, L. A. Knodler, and O. Steele-Mortimer Modulation and Utilization of Host Cell Phosphoinositides by Salmonella spp. Infect. Immun., August 1, 2004; 72(8): 4331 - 4335. [Full Text] [PDF]
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S. Tsunawaki, L. S. Yoshida, S. Nishida, T. Kobayashi, and T. Shimoyama Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase Infect. Immun., June 1, 2004; 72(6): 3373 - 3382. [Abstract] [Full Text] [PDF]
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K.-i. Uchiya, E. A. Groisman, and T. Nikai Involvement of Salmonella Pathogenicity Island 2 in the Up-Regulation of Interleukin-10 Expression in Macrophages: Role of Protein Kinase A Signal Pathway Infect. Immun., April 1, 2004; 72(4): 1964 - 1973. [Abstract] [Full Text] [PDF]
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J. D. Boddicker and B. D. Jones Lon Protease Activity Causes Down-Regulation of Salmonella Pathogenicity Island 1 Invasion Gene Expression after Infection of Epithelial Cells Infect. Immun., April 1, 2004; 72(4): 2002 - 2013. [Abstract] [Full Text] [PDF]
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T. van der Straaten, L. Zulianello, A. van Diepen, D. L. Granger, R. Janssen, and J. T. van Dissel Salmonella enterica Serovar Typhimurium RamA, Intracellular Oxidative Stress Response, and Bacterial Virulence Infect. Immun., February 1, 2004; 72(2): 996 - 1003. [Abstract] [Full Text] [PDF]
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D. M. Catron, Y. Lange, J. Borensztajn, M. D. Sylvester, B. D. Jones, and K. Haldar Salmonella enterica Serovar Typhimurium Requires Nonsterol Precursors of the Cholesterol Biosynthetic Pathway for Intracellular Proliferation Infect. Immun., February 1, 2004; 72(2): 1036 - 1042. [Abstract] [Full Text] [PDF]
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A. J. Roe, H. Yull, S. W. Naylor, M. J. Woodward, D. G. E. Smith, and D. L. Gally Heterogeneous Surface Expression of EspA Translocon Filaments by Escherichia coli O157:H7 Is Controlled at the Posttranscriptional Level Infect. Immun., October 1, 2003; 71(10): 5900 - 5909. [Abstract] [Full Text] [PDF]
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J. A. Carlyon, W.-T. Chan, J. Galan, D. Roos, and E. Fikrig Repression of rac2 mRNA Expression by Anaplasma phagocytophila Is Essential to the Inhibition of Superoxide Production and Bacterial Proliferation J. Immunol., December 15, 2002; 169(12): 7009 - 7018. [Abstract] [Full Text] [PDF]
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S. H. Browne, M. L. Lesnick, and D. G. Guiney Genetic Requirements for Salmonella-Induced Cytopathology in Human Monocyte-Derived Macrophages Infect. Immun., December 1, 2002; 70(12): 7126 - 7135. [Abstract] [Full Text] [PDF]
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J. H. Brumell, P. Tang, M. L. Zaharik, and B. B. Finlay Disruption of the Salmonella-Containing Vacuole Leads to Increased Replication of Salmonella enterica Serovar Typhimurium in the Cytosol of Epithelial Cells Infect. Immun., June 1, 2002; 70(6): 3264 - 3270. [Abstract] [Full Text] [PDF]
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A. van Diepen, T. van der Straaten, S. M. Holland, R. Janssen, and J. T. van Dissel A Superoxide-Hypersusceptible Salmonella enterica Serovar Typhimurium Mutant Is Attenuated but Regains Virulence in p47phox-/- Mice Infect. Immun., May 1, 2002; 70(5): 2614 - 2621. [Abstract] [Full Text] [PDF]
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A. Sansone, P. R. Watson, T. S. Wallis, P. R. Langford, and J. S. Kroll The role of two periplasmic copper- and zinc-cofactored superoxide dismutases in the virulence of Salmonella choleraesuis Microbiology, March 1, 2002; 148(3): 719 - 726. [Abstract] [Full Text] [PDF]
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D. Chakravortty, I. Hansen-Wester, and M. Hensel Salmonella Pathogenicity Island 2 Mediates Protection of Intracellular Salmonella from Reactive Nitrogen Intermediates J. Exp. Med., May 6, 2002; 195(9): 1155 - 1166. [Abstract] [Full Text] [PDF]
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p = 0.02 (n =
3).
