David Blanco, PhD MIMG C106
CHS A2-087G
Salmonella
Salmonellae are widely dispersed in nature, being found in the gastrointestinal
tracts of domesticated and wild animals, reptiles, birds, and insects. Salmonella
are commensals as well as pathogens that cause a spectrum of diseases in
humans and other animals. Some Salmonella species, such as S. typhi, S.
paratyphi, and S. sendai, are highly adapted to humans and have no other
known natural hosts. Others, such a S. typhimunrium, have a broad host range
infecting a variety of animals including humans. The species most studied in the
laboratory are S. typhi, S. typhimurium, S enteriditis, and S. cholerasuis. S.
enteriditis is one of the most common causes of food-borne gastroenteritis, a
self-limiting disease in humans. Because many farm animals carry S enteriditis
in their intestinal tracts, slaughterhouse byproducts are heavily contaminated.
Chicken and turkey parts, and the surface of eggs, are usually contaminated with
this organism, which is why it is important to wash poultry before cooking and to
never undercook poultry products. S. typhi is a specific human pathogen that
causes the serious, systemic, febrile, and life-threatening illness known as
typhoid fever. It is usually acquired by ingestion of food or water that has been
contaminated with human feces. S. typhimurium cause a self-limiting, localized
infection in humans but a systemic infection resembling typhoid fever in mice.
For this reason, S. typhimurium infection of mice has been studied extensively as
a model for human typhoid fever.
Following ingestion, Salmonella that survive the acidic environment of the
stomach and reach the small intestine preferentially invade M cells in the follicle-
associated epithelium (FAE) of Peyer’s patches. Destruction of M cells quickly
follows, forming a gap in the FAE which allows Salmonella to invade adjacent
enterocytes. This damage also allows Salmonella to gain access to the
reticuloendothelial system (RES), where they encounter macrophages. Data,
which we will discuss below, indicates that when Salmonella invades activated
marophages it stimulates these macrophages to undergo apoptosis
(programmed cell death). In contrast, if Salmonella invades a resting
macrophage (non-activated) it results in altered membrane trafficking and
prevention of phagosome-lysosome fusion. Survival of Salmonella in non-
activated macrophages facilitates bacterial spread to the spleen, liver, and bone
marrow.
PART I: How do Salmonella invade mammalian cells?
Invasion of polarized Madin-Darby Canine Kidney (MDCK) cells by Salmonella
has been used as an in vitro model to study the molecular mechanisms involved
in invasion. MDCK cells can be grown as a polarized monolayer where the cells
are joined by “tight junctions” and the apical surface displays microvilli, like
epithelial cells lining the G.I. track. When Salmonella are added to monolayers of
MDCK cells, the surface of the MDCK cells changes shape within 15 minutes,
and hair-like appendages, called invasomes, form. Within the next 15 minutes,
the invasomes disappear, and the MDCK cells change their morphology in the
region just beneath the bacteria: large cup-like membrane ruffles form,
enveloping the bacteria which are then internalized. Once inside, the bacteria
multiply within endosomes and eventually are released from the basolateral
surface of the MDCK cells.
This remarkable process requires genes within the Salmonella inv locus: Stains
with mutations in invG or invC never form invasomes and do not become
internalized; strains with mutations in invA or invE form invasomes but never
retract them and never become internalized.
How do the inv genes relate to invasomes and to invasion?
The inv locus is part of a 35 kb stretch of DNA at centisome 63 on the Salmonella
chromosome. Many additional loci required for invasion are also located here.
This region constitutes a pathogenicity island, called SPI1 (Salmonella
pathogenicity island 1). Several features of pathogenicity islands support the
model that they represent mobile genetic elements that confer pathogenicity
phenotypes:
Carry (often many) virulence related genes.
Presence in pathogenic strains, absence or sporadic distribution in less-
pathogenic strains of the same species.
Different G+C content compared to the rest of the chromosome.
Occupy large chromosomal regions (>30 kb).
Represent distinct genetic units.
Associated with tRNA genes and/or IS elements at boundaries.
Presence of (often cryptic) “mobility” genes.
Instability.
The Enteropathogenic Escherichia coli (EPEC) locus of enterocyte effacement
(LEE) is an example of how virulence phenotypes can be gained and lost by
acquisition of pathogenicity islands.
Enteropathogenic E. coli (EPEC) is a potentially fatal diarrhoeal pathogen that
strikes infants in developing countries. EPEC causes attaching and effacing
lesions when it interacts with intestinal epithelial cells – basically, microvilli are
replace by compact microfilamentous structures that look like pedestals beneath
the bacteria. Coincident with pedestal formation is the induction of host signal
transduction cascades that lead ultimately to diarrhea. Many genes required for
EPEC virulence were found to map to the LEE pathogenicity island.
To test the hypothesis that the LEE is a function cassette, sufficient to confer
upon non-pathogenic E. coli the ability to induce AE lesions, McDaniel and Kaper
(1997, Molecular Microbiology, 23:399-407):
Cloned the LEE locus into a plasmid.
Introduced this plasmid into several non-pathogenic lab strains of E. coli
such as DH5α and HB101.
Showed that DH5α and HB101 carrying the LEE-containing plasmid, but
not the plasmid vector alone, secreted a protein, EspB, that was
previously shown to be required for virulence.
Showed that DH5α and HB101 carrying the LEE-containing plasmid, but
not the plasmid vector alone, induced tyrosine phosphorylation of a
protein, HP-90, present in the host cell plasma membrane – a phenotype
previously shown to be associated with AE lesion formation.
Showed microscopically that DH5α and HB101 carrying the LEE-
containing plasmid, but not the plasmid vector alone, could induce the
formation of AE lesions in CaCo-2 cells.
These experiments provide strong evidence that specific aspects of pathogenicity
can be acquired by horizontal gene transfer of genetic elements such as
pathogenicity islands.
How do the Salmonella inv genes mediate invasion?
Many of the gene products encoded within the Salmonella SPI1 were found to be
similar to gene products that form type III or contact-dependent secretion
systems in Yersinia and Shigella.
Since these genes appear to encode a type III secretion system, are required for
invasion, and are required for the formation of invasomes, Jorge Galan’s group
hypothesized that the invasome surface appendages are the salmonella type III
secretion apparatus.
To test this hypothesis, they isolated a supramolecular structure from the
membranes of Salmonella typhimurium that appear to be at least part of the
secretion apparatus (Kubori et al, 1998 Science, 280:602).
They started with an flhC mutant of Salmonella. flhC is part of the master
regulatory locus controlling motility in Salmonella. Since proteins that form
the basal body of flagella are similar to components of type III secretion
systems, it was important to use a strain that could not produce flagella.
They osmotically shocked this strain and looked at the cytoplasmic and
outer membranes by trasmission electron microscopy (TEM). What was
observed were complex structures, which they called needle complexes,
that spanned the cytoplasmic and outer membranes and extended
outward from the cell surface.
They then isolated these needle complexes by CsCl density
centrifugation. The needle complexes were present in the flhC mutant,
but not in strains that also contained mutations in invG, prgH, or prgK.
They next separated the proteins in the purified needle complexes by
SDS-PAGE and stained them by silver stain. Proteins of 62-, 52-, and 31-
kDa were prominent. These are the predicted M.W. protein sizes (based
upon the deduced amino acid sequence derived from the gene sequence)
of InvG, PrgH, and PrgK.
They next constructed a strain expressing an epitope-tagged PrgH. The
epitope they used, called M45, could be detected by a specific monoclonal
antibody (anti-M45 MAb). The strain expressing the epitope tagged PrgH
could secrete proteins normally secreted by wild type Salmonella,
indicating that the epitope-tagged PrgH could form a functional secretion
apparatus. Needle complexes purified from the epitope-tagged PrgH
expressing strain were then incubated with anti-M45 MAb and visualized
by immunoelectron microscopy. Antibody molecules could be seen
around the basal structure as detected by an anti-mouse antibody
conjugated to the electron dense material colloidal gold.
These data suggest that the needle complexes are in fact the type III secretion
apparatus encoded by SPI1 and that they form the invasomes that mediate entry
into host cells.
What is the host cell contribution to Salmonella type III secretion mediated
invasion?
Interaction of Salmonella with eukaryotic cells induces profound rearrangement
of the eukaryotic cell cytoskeleton, the formation of membrane ruffles, and
subsequent internalization of the bacteria.
The induction of membrane ruffles is critical for entry of Salmonella:
mutants unable to induce ruffles do not invade.
The appearance of membrane ruffles is accompanied by profound
cytoskeletal rearrangements at the point of the bacterial-host cell contact
and several cytoskeletal proteins, including actin, αactin, talin, tubulin,
tropomyosin, and ezrin accumulate at these sites.
Salmonella infection of cultured cells is accompanied by a marked
increase in intracellular Ca2+ concentration: i) Ca2+ chelators and
antagonist of Ca2+ channels block entry of Salmonella and ii) mutants that
are unable to invade do not induce increases in intracellular Ca 2+
concentration.
Several actin-bundling proteins, such as gelsolin, villin, and plastin, can
become actin-serving proteins in response to increased intracellular Ca2+
concentration.
Membrane ruffling, cytoskeletal rearrangements, and Ca2+ fluxes have
been observed as a consequence of the activation of a number of host cell
surface receptors, including the epidermal growth factor receptor (EGFR).
Galan’s group has shown that infection of Henle-407 cells by Salmonella
is accompanied by activation of the EGFR.
Model for how bacterial attachment leads to internalization:
Attachment of Salmonella to the mammalian cell surface stimulates a
receptor (possibly the EGFR; epithelial growth factor receptor) that
initiates a signaling cascade resulting in the activation of MAP-kinase, a
central regulatory molecule.
MAP-kinase activates phopholipase A2 (PLA2).
Arachidonic acid (AA) is produced and converted to leukotriene D4 (LTD4).
LTD4 activates a calcium channel allowing the influx of Ca2+.
Increased [Ca2+] causes depolymerization of actin microfilaments that in
turn cause membrane blebs.
Release of profiling from the membrane can also participate in
cytoskeletal reorganization.
Supporting evidence for this model:
In Henle-407 cells, Salmonella infection results in stimulation of the EGFR.
invA mutants DO NOT stimulate the EGFR and DO NOT invade.
invA mutants +EGF result in stimulation of the EGFR and invasion of the
Salmonella.
E. coli (a nonpathogenic lab strain) +EGF results in stimulation of the
EGFR and internalization of the E. coli.
Inhibition of PLA2 or 5-LO prevents invasion.
Contact of Salmonella with Henle-407 cells results in increased LTD4
levels and increased [Ca2+].
Inv mutants + LTD4 cause increased [Ca2+] and invasion.
Chelation of extracellular Ca2+ or addition of Ca2+ channel antagonists
causes decreased ruffling and decreased invasion.
PART II: Interaction of Salmonella with macrophages
Salmonella induced apoptosis in (activated macrophages).
Salmonella are able to survive within macrophages and this ability has been
shown by several laboratories to be an important aspect of Salmonella
pathogenesis. In 1996 and 1997, however, four separate groups reported that
the interaction between Salmonella and macrophages in vitro sometimes results
in cytotoxicity and death of the macrophages.
For example, Chen et al. (1996, Molec. Microbiol. 21:1101) incubated wild type
Salmonella typhimurium with J774A.1 cells, a mouse macrophage-like cell line
routinely used for in vitro studies, and observed the interaction over time using
video microscopy. Within 2 to 15 minutes, increased membrane ruffling and
macropinocytosis in the macrophages was seen. From 15 to 30 minutes post-
inoculation, the ruffling and macropinocytosis decreased and after 30 minutes,
the J774A.1 cells began rounding up and lifting off the bottom of the tissue
culture wells. When macrophage cell death was quantitated by microscopic
examination of cells stained with the membrane-impermeant dye eithidium
homodimer-1, it was found that the percentage of dead cells increased from
about 20% at 20 minutes to about 80% at 8 hours post-inoculation.
Is the SPI1-encoded type III secretion system required for cytotoxicity?
To determine if the SPI1 encoded type III secretion system is involved in the
induction of macrophage cytotoxicity, Chen et al. compared cytotoxicity induced
by wild type Salmonella with that induced by strains with mutations in various
SPI1 loci. All mutants that were unable to invade tissue culture cells were also
unable to induce cytotoxicity in J774A.1 cells, while mutants that were not
defective for internalization induced cytotoxicity just like wild type Salmonella.
Therefore, type III secretion-dependent internalization of Salmonella coincided
with cytotoxicity.
Salmonella-induced cytotoxicity exhibits features of apoptosis
(programmed cell death).
Apoptosis – or programmed cell death – is the result of a highly regulated
process that is controlled by complex signal transduction pathways. In contrast
to necrotic death, such as when cells die from damage or poisoning and spill
their contents over neighboring cell eliciting an inflammatory response, in
apoptotic death the cell nucleus becomes condensed, the cell shrivel, and the
shrunken corpse is rapidly engulfed and digested by neighboring cells.
Apoptosis is an intrinsic part of embryogenesis, tissue turnover, immunological
killing mechanisms, and also of mechanisms of tumor growth and regression,
and apoptosis does not result in a host inflammatory reaction.
Chen et al. used three approaches to determine if macrophage cytotoxicity
induced by Salmonella occurred via apoptosis:
1. J774A.1 cells incubated with wild type Salmonella were stained with DAPI,
which stains DNA, and examined microscopically. Condensed and
fragmented chromatin, consistent with apoptotic death was observed.
2. J774A.1 cells incubated with wild type Salmonella were examined by
transmission electron microscopy: membrane blebbing, chromatin
condensation, and the presence of apoptotic bodies were seen.
3. Fragmented DNA in J774A.1 cell incubated with wild type Salmonella was
detected using a fluorescent TUNEL assay (TUNEL = terminal
deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end
labeling). The nuclei of J774A.1 cells infected with wild type Salmonella
stained positively in this assay.
Taken together, these data suggests Salmonella-induced death of J774A.1 cells
occurs by apoptosis.
Is bacterial internalization required for induction of apoptosis?
Salmonella strains with mutations in genes required for internalization did not
induce cytotoxicity while stains with mutations in SPI1 genes that were not
required for internalization did induce cytotoxicity. These results suggest that
internalization of Salmonella may be required for induction of
cytotoxicity/apoptosis. To test this hypothesis, Chen et al. incubated J774A.1
macrophages with Salmonella in the presence and absence of cytochalasin D.
Cytochalasin D disrupts the actin cytoskeleton preventing phagocytosis. In the
presensce of cytochalasin D therefore, Salmonella will adhere to the J774A.1
cells but will not be internalized. Chen et al. found that Salmonella induced
cytotoxicity in both the presence and absence of cytochalasin D (unlike Shigella,
which caused cytotoxicity only in the absence of cytochalasin D), suggesting that
Salmonella could trigger apoptosis from the cell surface. Since type III secretion
systems have been shown to be able to secrete and translocate proteins directly
into the cytoplasm, or plasma membranes of eukarytotic cells, this result
suggests one or more proteins secreted by the SPI1 encoded type III system
may be responsible for inducing apoptosis in host cells.
Does the “activation state” of the macrophage influence the induction of
apoptosis?
Monack et al. (1996, PNAS 93:9833) measured apoptosis induced by wild type
and several mutant strains of Salmonella in RAW264.7 macrophage-like cells
and bone marrow derive macrophages (BMM). The Salmonella mutants fell into
two classes: 1) those that could invade but could not replicate within
phagosomes (inv+ rep-) and 2) those that could not induce membrane fuffling
and could not invade, but could replicate in phagosomes (inv- rep+).
Three questions were addressed:
1. Is replication within the phagosome required for induction of apoptosis?
2. Is invasion required for induction of apoptosis?
3. Does the source (possibly reflecting the activation state) of the
macrophage influence the ability of Salmonella to induce apoptosis?
The ability of the [inv+ rep-] mutants to induce apoptosis in both RAW264.7 and
BMM macrophages was the same as wild type Salmonella, indicating the ability
to replicate within phagosomes is NOT required for induction of apoptosis. [inv-
rep+] mutants were unable to induce significant apoptosis in either type of
macrophage, suggesting invasion, or at least the ability to induce membrane
ruffles, is required to induce apoptosis. Wild type Salmonella (and the inv+ rep-
mutants) induced significantly more apoptosis in BMM than RAW264.7 cells,
suggesting that the “activation state” of the macrophage, which is probably
greater in BMM than in the RAW264.7 macrophage-like cell line, may influence
the ability of Salmonella to induce apoptosis. As mentioned earlier, Salmonella
does not always induce apoptosis in macrophages. In fact, the ability of
Salmonella to survive within macrophages was recognized as an important
aspect of Salmonella pathogenesis long before its ability to induce apoptosis was
discovered. One theory is that Salmonella either enter and survive within
macrophages or induce apoptosis depending on the activation state of the
macrophage. Salmonella may induce apoptosis in activated macrophages to
avoid from being killed themselves, i.e. kill or be killed. In contrast, Salmonella
may opt to invade and survive in resting macrophages so that they can be
transported to the liver, spleen, and bone marrow.
How are Salmonella able to survive within macrophages?
Expression of many virulence genes in Salmonella is controlled, at least in part,
by a two-component signal transduction system called PhoP/PhoQ. PhoQ is a
cytoplasmic membrane sensor molecule that responds to Mg2+ and Ca2+. In the
relative absence of Mg2+ and Ca2+, PhoQ is active, autophosphorylates at a
conserved histidine residue in its cytoplasmically located transmitter domain, and
transfers this phosphoryl group to PhoP. PhoP is a cytoplasmic response
regulator molecule that activates and/or represses gene transcription when
active. PhoP/PhoQ control the expression of two classes of genes: prg (Pho-
repressed genes) loci and pag (Pho-activated genes) loci. When PhoP/PhoQ is
inactive, in the presence of millimolar concentrations of Mg2+ or Ca2+, pag loci are
not expressed and prg loci are expressed. Prg loci include components of the
SPI1 encoded type III secretion system. The functions of pag loci are mostly
unknown.
To determine if either pag loci, prg loci, or both, are required for virulence, strains
that are either rendered PhoP/PhoQ inactive (PhoP- or PhoQ-) or constitutively
active (PhoPc) were constructed and compared with wild type Salmonella for
their ability to cause a lethal infection of mice following i.p. injection. Less than
20 wild type Salmonella are required to kill a mouse when administered by this
route. In contrast, the LD50s for PhoP-, PhoQ-, and PhoPc strains were about 105
(a 4 log increase in LD50). This result indicates that both pag and prg loci are
required to virulence. One particular pag gene knockout mutation, pagC, was
shown to also have an increased LD50.
To determine if pag loci, prg loci, or both, are required for the bacterial to survive
within macrophages, the “macrophage survival indexes” (MSI) for each of the
same strains were determined. MSI = the mean bacterial count at 24 hours post-
inoculation of macrophages / the mean bacterial count a 1 hour post-inoculation.
>1 = multiplication of the bacteria within the macrophages, <1 = death of the
bacteria. Data are on a log scale.
The MSI for wild type Salmonella was 6.13, indicating their ability to survive in
the macrophages. The MSI for all of the mutants tested were <1, indicating that
both pag and prg loci are required for survival of Salmonella.
How can both pag loci AND prg loci be required?
Clues came from looking at the course of events that occur following
internalization.
Pag gene expression is induced within macrophages, but not within epithelial
cells.
Sam Miller’s group (1992 PNAS 89:10079) constructed strains with lacZY fusions
to pag loci, or to a non-PhoP/PhoQ regulated gene (ibg) as a control. They
incubated macrophages with these strains then measured β-galactosidase
activity at 1, 4, and 6 hours post-internalization. For pag-lacZY containing
strains, β-galactosidase activity increased over time, while for the ibg-lacZY
containing strain, β-galactosidase activity remained constant. In CaCo-2 cells,
an epithelial cell line, β-galactosidase activity did not increase after
internalization.
Macrophages are equipped with several different mechanisms to destroy
microorganisms within phagosomes, including toxic oxygen derivatives, reactive
nitrogen intermediates, nutrient limitation, acidification of the compartment, and
fusion of phagosomes with lysosomes that are rich in degradative enzymes.
The observation that pag gene expression increased at 4 and 6 hours post-
internalization suggested a coincident change in the phagosome environment.
To test the possibility that pag gene expression increased in response to a drop
in pH, strains containing pag-lacZY fusions were grown in buffered L broth at
different pHs and β-galactosidase activity was measured. Pag-lacZ expression
was higher in cells grown at pH < 4.7 compared to cells that were grown at pH
5.0, indicating that pag gene expression is indeed regulated by pH.
To determine if the pag gene expression at 4 and 6 hours post-internalization in
macrophages was coincident with, and dependent on, acidifcation of the
phagosome, NH4Cl or chloroquine was added to the tissue culture medium.
These compounds prevent adicification of the phagosome. In the presence of
these compounds, pag-lacZ expression did NOT increase following
internalization. These data suggest that PhoP/PhoQ becomes active within the
phagocytic vesicle and that this activation coincides with a drop in pH.
(Remember, however, that in vitro, PhoP/PhoQ responds to Mg2+ and Ca2+. The
true signals sensed in nature by PhoP/PhoQ have not yet been definitively
proven. The change in pH within phagosomes could be coincidental).
Salmonella alters acidification of the phagosome.
Phagocytic vesicles containing yeast particles or latex beads have been shown
to become acidified within about 2 hours after phagocytosis. In the above
experiment, pag gene expression did not increase until about 4 hours post-
internalization. To determine if Salmonella actively influenced phagosome
acidification, the pH of phagosomes was measured after internalization of either
live or heat killed Salmonella. Phagosome pH was measured using FITC-
dextran. The fluorescence spectrum of fluorescein varies as a function of pH.
This was measured in individual phagosomes by quantitative fluorescence
microscopy. When heat-killed Salmonella were internalized, the pH of the
phagosome dropped rapidly to < 4.4 within about 1 hour. In contrast, when live
Salmonella were internalized, the pH of the phagosomes droped only to about 5
and only after 5 hours. So, live Salmonella apparently delayed and decreased
the magnitude of phagosome acidification.
Salmonella stimulate macrophage macropinocytosis.
How does Salmonella survive inside the early phagosome long enough to allow
delayed pag expression? (pag genes are required for survival within
phagosomes but are not induced until 4 to 6 hours post-internalization).
1. Salmonella stimulate macropinocytosis and persist in spacious
phagosomes.
2. PhoPc mutants were deficient in induction of spacious phagosomes.
These results suggest that prg-encoded proteins are involved in stimulation of
macropinocytosis and spacious phagosome formation.
Salmonella stimulate phagosomes to diverge from degradative endocytic
pathway.
Phagosomes destined to traffic along the degradative pathway of the host cell’s
endocytic network are thought to undergo a series of sequential biochemical
modifications and vesicle fusion reactions. A number of markers specifc for
unique compartments within the endocytic pathway have been used to
characterize changes that occur during the process. As the phagosome
matures, proteins which initially compose the newly formed compartment
disappear and are replaced, first by proteins present in early endosomes, such
as rab5, the transferring receptor, and low-density lipoproteins, and later by
proteins inherent to late endosomes/relysomes, including rab7, rab9, mannose-6-
phosphate receptors (M6PRs), and lysosomal membrane glycoproteins (LAMPs).
The function of M6PRs is to deliver a subset of newly synthesized, soluble,
lysosomal enzymes from the trans-Golgi network to the prelysosomal
compartments. The prelysosome is transformed into a degradative compartment
through the further incorporation and/or processing of lysosomal hydrolases such
as cathepsins, lysosomal acid phosphatases (LAP), and β-glucuronidase.
So, M6PR serves as a marker for fusion of late endosomes (lysosome) with the
phagosome, and markers delivered by M6PR-dependent mechanisms include
cathepsin-L. LAMP and LAP, markers for late endosome and lysosome fusion,
respectively, are delivered by M6PR-independent mechanisms.
To determine if Salmonella alters the trafficking pattern of phagosomes within
which it resides, Rathman et al. (1997, I&I 65:1475-1485) incubated
macrophages with either latex beads, wild type Salmonella or an inv- Salmonella
mutant for 30 minutes or 10 hours, stained with fluorescently labeled antibodies
against M6PR, LAMP1, LAP, and cathepsin, and examined them using
fluorescence microscopy. (Latex beads accumulated with all of these markers
over time, indicating the noramal trafficking of these phagosomes down an
endocytic pathway). By comparison, Salmonella containing phagosomes co-
localized with LAMP1 and LAP, but not M6PR or cathepsin, suggesting these
phagosomes had diverged from the endocytic pathway. Since co-localization of
markers on phagosomes containing the inv- strain was similar to that of
phagosomes containing wild type Salmonella, invasion via the SPI1 encoded
type III secretion system is apparently not required for this phenotype. This
suggests that regardless of whether Salmonella enter phagocytes by
phagocytosis or bacterial (type III) mediated entry, they are able to alter the
phagosome trafficking pattern. The ability to alter the trafficking patten was,
however, dependent on the viability of the Salmonella, as phagosomes
containing heat killed bacteria contained M6PR, LAMP1, LAP, and cathepsin-L,
just like phagosomes containing latex beads.
Resistance to defensins is a pag-encoded phenotype.
Defensins are amphipathic, cationic, antimicrobial peptides (approx. 4-kDa in
size) found within neutrophil granules, macrophage phagosomes, and secretions
of mucosal epithelia. Defensins probably kill gram negative bacteria by binding
to LPS through ionic bonds with the unsubstituted, negatively charged
phosphoryl groups of lipid A.
Salmonella grown under conditions in which PhoP/PhoQ is active are relatively
resistant to a particular rabbit defensin called NP-1. PhoP- and PhoQ- strains
are sensitive to NP-1 while PhoPc mutants are resistant to NP-1.
Polymyxin B is a cationic antibiotic that kills bacteria by a mechanism similar to
that of defensins. Polymyxin B resistance in Salmonella requires the pmrA locus.
PmrAB was shown to be positively regulated by PhoP/PhoQ. To determine if
Polymyxin B resistance is controlled by PhoP/PhoQ, wild type and mutant strains
of Salmonella were compared for their ability to grow in the presence of different
concentrations of Polymyxin B. PhoP-, PmrA-, or wild type Salmonella grown
under conditions in which PhoP/PhoQ is inactive, were sensitive to low
concentrations of Polymyxin B, while PhoPc strains and wild type Salmonella
grown under conditions where PhoP/PhoQ is active were resistance to high
concentrations of Polymyxin B, indicating that like resistance to defensins, similar
resistance to Polymyxin B is pag-encoded.