- Email: [email protected]
In vivo and ex vivo regulation of bacterial virulence gene expression
17
of
regulation
In vivo and ex vivo expression
aacterlal
r--_--_,-l
vwlence
gene
__~-__I_---
_____
_
Peggy A Cotter* and Jeff F Millet-t Bacteria
are remarkably
to survive
adaptable
and multiply
environments. of genetic
Adaptability
information
mechanisms
conferring
particular
situation
deleterious
organisms
expression. while bacteria
within
host cells and tissues
Being
of little or no use to the bacterium
specific factors
stages
to appreciate bacteria
gene
the complexities
to this rule.
except
during
to tight and coordinate advances,
we are beginning
of the interactions
and their hosts. The ability to probe
regulation
gene
in viva has broadened
between
virulence
our perspectives
on
pathogenesis.
Addresses Department of Microbiology and Immunology, University of California at Los Angeles School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-l 747, USA *e-mail: [email protected] +e-mail: [email protected] Current
Opinion
in Microbiology
1998,
1 :17-26
http://biomednet.com/elecref/1369527400100017 0 Current Biology Ltd ISSN
1369-5274
Abbreviations CSP CT
competence stimulating peptide cholera toxin
DFI
differential fluorescence
FACS FAE GFP
fluorescence activated cell sorting follicle associated epithelium green fluorescent protein histidine phosphotransfer domain homoserine lactone in viva expression technology
HPt HSL IVET SPI
Salmonela
pathogenicity
STM
signature tagged
TCP
toxin co-regulated
hosts [Z]. Diffusible signals can the bacteria themselves, conferring cell density [3,4]. Finally, sensed by direct physical
also be produced by the ability to measure
host cells can apparently contact between bacteria
be and
target eukaryotic cells [So*]. Signals that affect virulence control systems in vitro can be used to effectively identify regulated genes; however, in many cases their relationship to signals important in viva is not well understood.
on and
cycle, these accessory
subject
As a result of recent
or
of virulence to multiply
gene
in a
unnecessary
are no exception
of the infectious
are nearly always
regulation.
advantage
are not. Expression
that allow pathogenic
and by the
In general,
or survival
are expressed,
functions
hostile
by the complement
to an organism
gene
a growth
that are able
and sometimes
is determined
available
that control
products
products
in diverse
induction
island
mutagenesis pilus
Introduction Our understanding of the mechanisms that govern bacterial virulence gene expression is relatively advanced compared with our understanding of when and why virulence gene regulation actually occurs. A diverse array of extracellular signals have been identified that modulate virulence gene expression in vitro. Many of these signals are general and seemingly nonspecific physical or chemical cues [ 1’1. In other cases bacteria have evolved to recognize specific metabolic products produced by their respective
Recognition that virulence loci are tightly and precisely regulated has spurred a flurry of activity towards identifying genes that are specifically expressed i?i viva. While this is an important goal, the ability to detect genes that are truly ‘in viva induced’ requires a detailed understanding of the relevant ex vlvo conditions. This in turn necessitates an appreciation of the entire infectious cycle and the array of environments that characterize it. Pathogenesis can be viewed as a developmental process characterized by a progression of spatially and temporally defined events. The most pertinent questions to ask regarding virulence gene control involve relative gene expression in relevant microenvironments. Virulence gene regulation is therefore best considered in a truly ecological context, and it is reasonable to expect that ecological complexity will be mirrored by the complexity of the regulatory circuitry that modulates gene expression. Our aim in this review is to focus on a selected set of virulence regulatory systems that illustrate general principles. Useful experimental approaches and results obtained in viva will be emphasized. Although we focus exclusively on pathogens of mammals, many of the same principles are apparent from studies of bacterial-plant interactions [6] and symbiotic
Virulence Virulence
interactions
with
invertebrate
hosts
[7].
regulatory components expression
is usually
governed
by
signaling
and regulatory mechanisms similar to those that control genes that are not specific to pathogenesis. One of the most regulatory
commonly encountered families of virulence proteins is the two-component group of signal
transducers. These couple a diverse array of signals to multiple effector outputs using regulated phosphotransfer reactions. Hallmarks of two-component systems are their modularity and accompanying adaptability. These features are highlighted by several recent developments. Three types of conserved signal transducing components are found in two-component systems. These are illustrated in Figure la using the Salmonella PhoQ/PhoP and Bordetella BvgS/BvgA virulence control systems as specific examples. In both cases signal transduction begins with autocatalyzed phosphorylation of a sensor kinase in the
18
Host-microbe
interactions: bacteria
presence of activating environmental conditions and ATP. This reaction is catalyzed by a transmitter domain which is linked to an input module with direct or indirect sensory capabilities. The input domains of PhoQ, BvgS and most other sensor proteins span the cytoplasmic membrane and they are generally assumed to control the autophosphorylation reaction. In vitro, PhoQ activity can be modulated by hlgz+ and CaZ+ [8] while BvgS responds to temperature, SOqz- and nicotinic acid [9’]. Sensor proteins serve as substrates for phosphocransfer reactions between transmitter phosphohistidines and conserved aspartic acid residues in receiver domains. For PhoQ/PhoP, this activates the output domain of the PhoP response regulator, allowing activation and repression of target gene transcription. In contrast, the BvgS/BvgA system operates by a more complex phosphorelay [9*,10’]. This signaling mechanism was initially discovered by Burbulys et al. [ll] in their elegant studies of Bacillus subtilis sporulation. As shown for BvgS, two receiver domains and a histidine phosphotransfer domain (Hpt) are involved in the phosphorelay. Phosphorylation of the BvgS receiver results in phosphoryl group transfer to water, an abortive event, or transfer to a conserved histidine in the Hpt. The phosphorylated Hpt is the substrate for phosphotransfer to the cognate receiver in the BvgA response regulator. This type of phosphorelay is widely distributed in nature [12]. The individual components can be connected in a variety of configurations or be contained on completely separate proteins. Why might a complex phosphorelay exist? PhoQ/PhoP and BvgS/BvgA perform apparently similar functions, but in the latter case twice as many phosphotransfer reactions are required. For any integrated circuit, the exact relationships between input and output will depend on the specifications of the components. hlultiple components with different properties could therefore fine-tune this relationship. An alternative but not mutually exclusive idea is that multiple steps allow multiple points for control. studies
This has received recent support, of sporulation. In B. subtih, proteins
again from that inhibit
autophosphorylation [13’] as well as receiver-specific phosphatases [14] have recently been discovered. It is also important to consider that two-component systems may not simply function as on/off switches for gene expression. This is especially apparent in the case of BvgAS [15]. In B. bromhiseptica, a respiratory pathogen of many fourlegged mammals, this system activates genes required for respiratory tract colonization and represses genes involved in motility. A class of BvgAS-regulated polypeptides expressed between these two extremes has recently been systems can discovered (Figure lb) [15]. T wo-component therefore control multiple waves of gene expression. An examination of available genomic sequences indicates that the number of two component family members present in a bacterium can vary dramatically [16]. For example, Helicobacterpylori appears to encode four sensors and six response regulators; Hemophihs ir@~enzae encodes four
sensors and coli encodes
seven response an estimated
regulators; 16 sensors
while Escherkhia and 40 response
regulators! It is intriguing to speculate that the number of two component family members encoded in a particular genome may correlate with the ecological complexity of the bacterium’s life cycle.
Hierarchical control and evolution of the Vibrio cholefae virulence regulon V&o dolerae is a motile, Gram-negative, curved rod which is endemic to ocean waters where it is either free-living or exists in association with copepods and other crustacea. When ingested by humans, this noninvasive pathogen causes severe secretory diarrhea. Adherence of bacteria to the intestinal epithelium is mediated in part by the toxin co-regulated pilus (TCP), and massive fluid loss results from the action of a potent ADP-ribosylating exotoxin (cholera toxin, CT). CT and TCP expression has been known for many years to respond to temperature, pH and other environmental conditions [17]. Early studies by Miller and hlekalanos [18] identified the toxRto.vS regulatory locus, which controls CT and TCP expression as well as the expression of more than 20 additional genes. Subsequent genetic and biochemical analyses contributed to the elucidation of the regulatory hierarchy shown in Figure 2 [17]. ToxR/ToxS control of virulence gene expression is mediated largely via ToxT, and it appears that differential expression of CT and TCP in the V cholerae classical and El Tor biotypes stems from differences in ToxR/ToxS-mediated induction of toxT expression [ 191 which may actually occur via TcpP/TcpH [ZO]. Genes within the ToxT-dependent pathway encode accessory colonization factors (a@, additional regulatory loci (tcpZ, tcpP and tcpH), cholera toxin &A and ctxB), TCP (tcpA-F) and toxT itself. Other genes, such as ompU and ompT, appear to be directly regulated by ToxR/ToxS. This hierarchically organized regulatory circuit may reflect a need to integrate various signal inputs to maintain separate but coordinated control of multiple genes. Two recent observations provide insights regarding the evolution of the ToxR regulon. The first is the recognition by Kovach et a/. [Zl] that the linked tcp, toxT and ad loci reside on a large pathogenicity island. The second is the discovery by Waldor and hlekalanos [22*] that OXA and ctxB are encoded on a lysogenic filamentous bacteriophage designated CTX@. These observations suggest that the hierarchical structure of the ToxR regulon reflects the horizontal acquisition of genetic elements encoding determinants for pathogenesis in humans. It has been proposed that ToxR/ToxS originally evolved to control outer membrane protein synthesis in an ancestor lacking the tcp-acf pathogenicity island and CTX$ [23,24’]. In support of this hypothesis, ToxR/ToxS homologues are present in nontoxigenic, non-TCP-expressing 1,: dolerae strains [l&21]. ToxR/ToxS homologues are also present in Vibriofischeri and Photobactetiwn sp. strain SS9, marine microbes that are not associated with human disease
In viva and ex viva regulation of bacterial virulence gene expression
Cotter
and Miller
19
Figure 1
(a)
Environmental signals Transmitter
Input
Receiver
PhoQ
Environmental signals
Output
PhoP
4
Transmitter
Input
Receiver
Hpt
Receiver
BvgS
Output
BvgA
oBvg+
Bvgi
Bvg-
?
Flagella urease
2.
3.
A:‘,$,“;s 1.
Current Opinion on Microbiology The two-component
group
of signal transducers
regulate
(a) The Salmonella
gene expression.
transduction systems. histidine residue (Hl)
The input domains of PhoQ and BvgS sense environmental in the transmitter domain. The transmitter phosphohistidines
aspartic
(Dl)
acid residues
regulation
of target
in the receiver
gene transcription.
domains.
For Phoa/PhoP
The phosphorylation
phosphorylation
of the BvgS
Dl
present
2). and Bvgmodulation
in the input domains.
phase
by environmental
by the expression
(b) Relative expression
loci (line 3) are indicated
of adhesins
conditions. and toxins,
on the vertical
ln viva experiments is necessary
expression of motility and other co-regulated Bvgi phase expressed antigens is unknown.
phenotypes,
of Dl
receiver
histidine (H2) on the histidine phosphotransfer domain (Hpt). The phosphotylated conserved aspartic acid residue (D2) in the BvgA receiver domain; this activates sequences
[24*,25]. The fact that TCP provides the receptor for CTXQ, and that both the tcp-acf cluster and ctxAB are controlled by ToxT, suggests their co-evolution may have preceded their association with 1! cholerae. Once introduced into II dolerae, control of these genes may
in transfer
the output
domain
of a phosphoryl
loci (line l), Bvg intermediate
axis indicates
for respiratory
is advantageous
and Bordetella
BvgS/BvgA
sensory
of PhoP resulting group
in
to a conserved
Hpt serves as a substrate for phosphotransfer to the the BvgA output domain. TM corresponds to transmembrane
with B. bronchiseptica
and sufficient
activates
results
levels of Bvg+ phase
axis. The horizontal
PhoP/PhoQ
signals which result in autocatalyzed phosphorylation of a serve as substrates for phosphotransfer to the conserved
phase-locked tract
for growth
phase
the phase of the organism
infection,
mutants
indicate
while the Bvg-
under nutrient-limiting
loci (Bvgi) (line
which
results
the Bvg+ phase, phase,
conditions
from
characterized
characterized
by the
(151. The function(s)
of
have come under ToxR/ToxS control via ToxT, thus converting ToxR/ToxS into a regulator of virulence gene expression. Interestingly, since TCP serves as the receptor for CTX$, ToxR/ToxS has been converted into a regulator of horizontal gene transfer as well.
20
Host-microbe
interactions: bacteria
Figure 2
B ToxR
Outer
membrane
ToxS
inner membrane
TCP-ACF
The K cholerae ToxR/ToxS regulon. ToxR and ToxS are cytoplasmic membrane expression of ompT. ToxR/ToxS controls expression of the cholera toxin genes
proteins.
ToxR/ToxS activates expression of ompU and represses encoded on the bacteriophage CTXQ and
(ctuA and CM)
activates the expression of genes on the TCP-ACF pathogenicity island, including genes encoding the toxin co-regulated pilus (tcp genes). ToxR/ToxS may control expression of accessory colonization factors (acf genes) indirectly via ToxT. It appears that TcpP/TcpH, which are also predicted to be cytoplasmic membrane proteins, activates expression of toxT, and ToxT activates tcp, acf, and ctu loci [20,211. For simplicity, regulatory
functions
of fcp loci are not shown.
Recognition that the primary virulence determinants of V cholerae are contained on discrete genetic elements suggests that horizontal gene transfer provides a mechanism for converting a nonpathogenic marine microbe into a pathogen responsible for severe human disease. It is also interesting to speculate on the function of the virulence regulatory system throughout the infectious cycle. ToxR/ToxS-activated genes are certainly required in viva, but they may also provide unrecognized advantages within the marine environment. A complete analysis of virulence gene expression by this important bacterium will require model systems that reflect the ecological niches characterizing
the entire
infectious
cycle.
Quorum sensing Quorum sensing differs from other signal-dependent responses in two major respects: the signal is produced by the bacteria themselves and the regulatory system measures bacterial cell density and growth phase. Several Gram-negative bacteria produce N-acyl homoserine lactone (HSL) molecules which serve as membrane-permeable signals. HSLs accumulate during growth and activate gene expression by binding to cytoplasmic receptor proteins that function as HSL-dependent activators [3]. Transcriptional activation is highly dependent on HSL concentration. This type of quorum sensing is found in a
variety of Gram-negative bacteria that establish symbiotic or pathogenic associations with plants or animals. In the opportunistic human pathogen Pseudomonas aeruginosa, two quorum-sensing systems (/as and I-I?/) regulate virulence gene expression [26,27]. Quorum sensing in Gram-positive bacteria follows a distinctly different paradigm [4]. The signaling molecule is usually a post-translationally processed, secreted peptide pheromone, which is sensed by the input domain of a two-component sensor. For Staphylococcus aureus, it has recently become apparent that multiple virulence factors are coordinately regulated by the quorum-sensing system summarized in Figure 3 [28]. Most of the signaling components are encoded by the agrBDCA operon. The agrD product is processed to yield a 7-9 amino acid peptide which is secreted into the culture supernatant. Pheromone production is dependent on AgrB, but the exact role of this protein is unclear. During post-exponential phase growth in vitro, the concentration of pheromone reaches a critical threshold level sufficient for activating AgrC, a transmitter-containing sensor protein with multiple transmembrane sequences. Phosphorylation of the AgrA response regulator apparently leads to transcriptional activation of two divergent promoters. The P2 promoter transcribes the agr operon itself, thereby amplifying the
In viva and ex viva regulation of bacterial virulence gene expression Cotter and Miller
sensory
response.
The
P3 promoter
initiates
expression
of a 0.5 kb transcript, RNAIII, which is the actual effector molecule. Although its mechanism of action is not yet clear, RNA111 appears to act primarily at the level of transcription of regulated genes. Genes encoding protein A, fibronectin-binding protein, coagulase and other surface proteins with potential roles in establishing infection and evading host defenses are preferentially expressed during exponential growth if1 vitro. This is before agr becomes active, and activated agr represses their transcription. Conversely, genes encoding a variety of excreted factors are induced when agr is active. These include alpha-hemolysin, enterotoxin B.
toxic shock
syndrome
toxin,
and
In a recent report by Ji et al. [29”], different strains of S. aureus were shown to produce structurally different peptide pheromones. Pheromones released by one strain could repress virulence gene expression in another. This ‘bacterial interference’ (so called because of the production of inhibitory peptides) could potentially exclude competing strains from occupying sites for colonization. The observation that exogenous peptides can inhibit staphylococcal virulence may lead to new therapeutic modalities for this and other Gram-positive pathogens. sensing is also used by Streptococcus regulate transformation competence and other phenotypes as well. Two separate two-component systems are involved in controlling transformation. The ComD/ComE proteins sense and respond to the competence-stimulating peptide (CSP) [30], and the ActR/ActH system is required for CSP production [31]. Mutation of actH also results in impaired nasopharyngeal colonization in rats, making ActR/ActH the first two-component system shown to be important for pneumococcal virulence [31]. Peptide-dependent
pneumoniae
to
What, exactly, is the linking virulence gene population or growth
selective advantage conferred by expression to the size of a bacterial phase? In the case of S. aureus,
the switch between the expression of surface proteins and excreted toxins may reflect a dichotomy between colonization and disease. Do greater numbers of bacteria provide an opportunity for host invasion? Does entry into post-exponential phase indicate impending bacterial doom? Perhaps the most important question is how to address the relevance of quorum sensing experimentally. It is clear that the agr regulon is needed for S. aureus virulence, indicating the importance of inducing exoprotein production. Less apparent is the need for repressing the expression of surface proteins during postexponential growth, and the reason why excreted factors are repressed in the exponential phase. An approach that might yield insights is to ‘rewire’ the control system to abrogate density-dependent regulation. Animal models that measure colonization, as compared with only disease, will be also required to understand the relationships between
cell density-
and growth-phase-dependent
regulation
21
and
pathogenesis.
The Salmonella paradigm: probing virulence gene expression in vi110 Sa/monel/a &Ohimutium causes
self-limiting gastroenteritis in humans. Infection of mice, however, results in a systemic illness similar to human typhoid fever. Following ingestion, S. typhutium 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 [32]. Destruction of the M cells quickly follows, forming a gap in the FAE which allows invasion of adjacent enterocytes. This damage also allows S. ~phmurium to gain access to the reticuloendothelial Invasion of activated macrophages appears to system. induce their apoptosis [33*,34] while invasion of resting macrophages results in altered membrane trafficking and prevention of phagosome-lysosome fusion. Survival of S. t)phimutium in nonactivated macrophages facilitates bacterial spread to the spleen, liver and bone marrow. The organism’s ability to survive in these various intracellular and extracellular environments requires a vast array of gene products. These are encoded on at least two pathogenicity islands, a virulence plasmid, and various linked and unlinked chromosomal loci. As seen with li chokrae, horizontally acquired genetic elements have apparently co-opted chromosomal control factors, resulting in a hierarchically organized complex regulatory network. Virulence genes in S. qphimurium appear to be controlled primarily in response to whether the bacterium is in an intracellular or extracellular compartment (Figure 4). Two regulatory systems of major importance are SirA/HilA and PhoP/PhoQ. Extracellular S. &Gimutium appear to be primed for invasion of a variety of host cell types. Invasion of M cells in viva and epithelial cells and macrophages in vitro requires a Type III secretion system encoded on the SPl Salmonella pathogenicity island located at centisome 63 [1*,35,36]. This Type III system triggers a complex cascade of signaling events in the host cell that results in marked cytoskeletal rearrangements, membrane ruffling and bacterial uptake by macropinocytosis. In Yersinia species, contact with host cells via a similar Type III secretion system results in the delivery of antiphagocytic proteins directly into the target cell cytoplasm rather than in internalization of the bacterium. By including a transcriptional repressor in the class of exported proteins,
lhinia contact
couple virulence gene [5**]. In S. Ilp/rimurium
regulation with genes encoding
host cell the SPIl
Type III system, and its secreted targets, are positively activated by HilA [37*]. Expression of hi/A was recently shown to require sirA, which encodes a two-component response regulator [38*], suggesting the existence of a cognate sensor protein that responds to cues in the extracellular milieu. Since the original sirA mutant could also be complemented by the unlinked loci sirB and sid, control of MA by sirA may not be direct.
22
Host-microbe
interactions: bacteria
Figure 3
S. aureus strains Interference
U
AgrB
P3
(?)
P2
agrA
agrC
a
AgrB
(?) a
RNAM R n Peptide 0 Protein
A
coagulase FBP
Capsule TSST a-hemolysin exotoxin
B Current Opmon in Microbiology
Quorum
sensing
in S. aureus [28,29”1.
of rnaiii and the agrBDCA pheromones are secreted
The agrSDCA
locus encodes
loci in response to the presence by ATP-binding cassette exporter
the AgrC/AgrA
either directly or indirectly, activates expression of capsule, toxic shock syndrome expression of protein A, coagulase and fibronectin-binding protein (FBP). Peptide producing
them can in some cases
inhibit
agr in other strains
two-component
regulatory
system
of peptide pheromones. In many cases, post-translationally proteins; however, such a factor has not been described
and cause
Following internalization by target cells, the PhoP/PhoQ sensory system becomes activated, possibly by recognizing differences in Caz+ and/or Mg2+ ion concentrations between extracellular and intracellular environments [S]. Activated PhoP simultaneously represses prgs (PhoP/PhoQrepressed genes), including those encoding the SPIl Type III secretion system, and activates expression of pags (PhoP/PhoQ-activated genes). The ability to survive in macrophages is crucial to Sa/mone//a pathogenesis and the requirement for expression of pag loci is well established in this regard (39,401. It was recently shown
expression
toxin (TSST), a-hemolysin and exotoxin B, and represses pheromones which activate the agr regulon in strains
‘interference’
that
that controls
processed peptide for the agr system. RNAIII,
[29”1.
macrophage survival results, at least in part, from and pmd expression. These are pags that encode yet another two-component regulatory system. PmrA/PmrB control resistance to cationic antimicrobial peptides such as those present in macrophage phagosomes [41,42]. Other intracellularly active regulatory factors include SlyA, which contributes to survival in macrophages and destruction of M cells [43,44], EnvZ/OmpR, which contributes to macrophage killing [45], and RpoS, which contributes to destruction of hl cells and spv-mediated growth in the liver and spleen [46]. Virulence genes expressed by intracellu-
pmfA
In viva and ex viva regulation of bacterial virulence gene expression Cotter
and Miller
23
Figure 4
I
Extracellularly
Invasion
of
I
located salmonella
orgA
prgKJIH
sipADCB
MA iagf
invCBAEGf
spa TSROPONM
invH @ I,
----------------
T
(E) [FEiF)&-s~Ra”,_
0
(TiE$gj [killing
j A
_fJ
P
S”
P
I
_J
-\
II
ssaUTSROPONVMLKJ
Y
V
PhoQ
EnvZ
SP12
(polymyxin6’)
OM
lntracellularly
located salmonella
Current Opmon
Virulence
gene regulation
in S. typhimurium
[35,36,37’,36’1.
The SirA/HilA
regulon,
which
is expressed
primarily
when
Salmonella
I” Mwobiology
are
extracellularly located (i.e. not found in host cells), is shown above the dotted line. Expression of hi/A requires SirA, a possible two-component response regulator. HilA positively activates the spa, inv and prg operons of the Salmone//a pathogenic&y island 1 (SPIl) which encode proteins that form the Type Ill secretion apparatus dotted genes
apparatus
and allow the invasion
(TIII). The Sip proteins,
of epithelial
whose
cells by S. typhimurium
expression
is activated
and the invasion
by Inf, are exported
and induction
of apoptosis
through
the Type Ill secretion
in macrophages.
Below
the
line, genes expressed primarily by intracellularly localized Salmonella are shown. PhoP/PhoQ represses expression of SPll -encoded (bar leading up to dotted line) and activates expression of genes and operons (i.e. s/yA and pag loci) required for intracellular survival.
Other regulons contributing to the phenotypes of intracellularly located S. fyphimurium are also shown. The function system encoded on SP12 is not known. The bacterial inner membrane (IM) and outer membrane (OM) are shown.
larly localized Salmonella, therefore, array of regulatory systems. In vivo expression
are controlled
by an
technology
Genetic tractability and the availability of 171 vitro and irr viva models for invasion and pathogenesis have made Salmonella an excellent system for developing methods to identify virulence genes and monitor their regulation. Ztr viva expression technology (WET) is a promoter trap scheme in which random DNA fragments are cloned in front of promoterless purA and /acZ genes such
of the Type Ill secretion
that genes that are expressed in vivo but not under specfic conditions if/ vitro can be identified (47). The assumption is that at least some virulence genes will conform to this overall pattern of expression. The IVET concept was a breakthrough and it has been successfully used with Salmonella, Pseudomonas, and Ye&zia species [48-SO]. The initial IVET system required sustained gene expression in animals. A recently developed version based on site-specific recombination in response to resolvase (tnpR) gene expression allows the identification of loci that are only transiently expressed i7z viva [51]. A potential
24
Host-microbe
disadvantage
requires j?~ vivo
interactions: bacteria
to the
IVET
approach
the proper choice induced promoters
is that
its success
of ex viva conditions, will only be detected
as if
the conditions chosen for growth in culture efficiently repress their expression. The original methodology was developed using mice versus agar as growth media. IVET’s maximum potential, however, may lie in identifying genes that are differentially expressed between two or more environments which are truly relevant to a pathogen’s infectious
cycle.
Signature-tagged
system [53,54*]. Although SPIZ-encoded genes appear not to be involved in invasion, survival or replication in macrophages, or induction of apoptosis, SPIZ mutants are severely attenuated after oral or intraperitoneal inoculation of mice [52,54’,55]. STM will undoubtedly lead to the discovery of previously unrecognized virulence genes in many bacterial pathogens. Green fluorescent protein technology The ultimate goal in understanding bacterial virulence gene regulation is to determine the dynamics of gene expression throughout the infectious cycle. Differential fluorescence induction (DFI) uses fluorescence-activated sorting (FACS) basis of their
to screen ability to
animals. It was shown that all of the promoters identified using macrophages it) vitro were expressed in splenocytes and hepatocytes following intraperitoneal inoculation of mice [57”]. This system represents a powerful new technology for identifying genes that are expressed in response to specific environments and for monitoring expression patterns during the process of pathogenesis.
Conclusions
mutagenesis
A more recent technology, termed signature-tagged mutagenesis (STM), is designed to identify genes that are required for growth or survival 171 v&o, but not for growth irr vitro [SZ]. SThl differs from IVET in that assumptions regarding gene expression are not required. STM provides a mechanism for identifying individual avirulent mutants in large pools of mutagenized bacteria, allowing a relatively small number of animals to be used for the initial screen. STM is a transposon-based mutagenesis scheme in which each transposon is marked with a different DNA sequence tag, permitting the comparison of mutants that survive passage through an animal with those that survive passage through culture medium. This powerful screening method led to the discovery of a second pathogenicity island in Salmonella, SPIZ, which appears to encode a Type III secretion
cell the
and at least one gene encoded on SPIZ were also detected in this screen. Because identified promoters are fused to gfp, expression can be monitored in tissues of infected
for active promoters drive expression of
on the
Aequorea Victoria jellyfish green fluorescent protein (GFP) [56]. DFI facilitates the selection of promoters that are differentially expressed in response to defined environmental conditions, including intracellular localization in eukaryotic cells. Major advantages of this approach include sensitivity and adaptability. Gene regulation can be observed at the level of individual bacterial cells, and variability in expression levels within a population can be determined. Bacteria, or eukaryotic cells containing them, can also be sorted on the basis of fluorescence intensity. Using DFI, Valdivia and Falkow [57”] identified several Salmonella promoters that were well expressed in macrophages yet poorly expressed in tissue culture medium alone [57”]. Nine of the 14 loci identified were regulated by PhoP/PhoQ. An EnvZ/OmpR-regulated gene
hluch of this discussion has focused on the adaptability of bacterial pathogens. The examples described above highlight another feature of these organismsnamely their remarkable diversity. Although similar components are often used to couple signals to responses, they are adapted by different bacteria to accomplish distinctly different tasks. An excellent example is the signaling modules that constitute two-component systems. They are used by S. aureus to sense other bacteria, by Salmonella species to differentiate extracellular from intracellular environments, and by B. bronchiseptica to sense whether they are inside a host or not. Type III systems are another example of similar components being used for significantly different reasons. Diversity can also be seen in the organization of virulence control networks. These regulatory circuits appear to be quite dynamic in an evolutionary sense, having the ability to adapt existing regulatory systems to control horizontally acquired genes. Over the last few years, our ability to probe virulence gene regulation has advanced considerably. Combining new methodologies with appropriate in vivo and ex viva models will highlight the diverse mechanisms pathogens use to adapt to their mammalian hosts.
Acknowledgements W
thank refcrcnced and unrcfcrenced cnllcagues for a wealth nf inrerexing expcrimenrs, data and insrghts, and hlingshun Liu for help with technical aspects of the review. The authors are supported by grants from the National Insrirute nf Health (grants A138417 and AI38955) and the American Cancer Socict~ (grant Ihl-791).
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . l
*
of special interest of outstanding interest
1. .
Finlay BB, Falkow S: Common themes in microbial pathogenicity revisited. Microbial MO/ Biol Rev 1997, 61:136i69. A broad review of the field of bacterial pathogenesis with an extensive reference list. 2.
Winans SC, Mantis NJ, Chen CY, Chang CH, Han DC: Host recognition by the VirA, VirG two-component regulatory proteins of agrobacterium tumefaciens. Res Microbial 1994, 145:461-473.
3.
Fuqua C, Winans SC, Greenberg EP: Census and consensus in bacterial ecosystems: the LuxR-Luxl family of quorum-sensing transcriptional regulators. Annu Rev Microbial 1996, 50:727751.
In viva and ex viva regulation
4.
Kleerebezem M, Quadri LE, Kuipers OP, de Vos WM: Quorum sensinq bv oeotide oheromones and two-comoonent sianaltransd&t& s&ten& in Gram-positive bacteria. MO/ Mi~obiol 1997, 24:895-904.
5. ..
Pettersson J, Nordfelth R, Dibinina E, Bergman T, Gustafsson M, Maonusson KE. Wolf-Watz H: Modulation of virulence factor exiression by’pathogen target cell contact. Science 1996, 273:1231-l 233. The first demonstration of regulation of virulence gene expression by contact with host cells. 6.
Long SR: Rhizobium symbiosis: f/ant Cell 1996, 8:1885-l 896.
7.
Ruby EG: Lessons from a cooperative, bacterial-animal association: the Vibrio fischeri-Euprymna scolopes light organ symbiosis. Annu Rev Microbial 1996, 50591-624.
8.
nod factors
in perspective.
Garcia-Vescovi E, Soncini FC, Groisman EA: Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence. Ce// 1996, 84:165-l 74.
Uhl MA, Miller JF: Integration of multiple domains in a twocomponent sensor protein: the Bordetella pertussis BvgAS phosphorelay. EMBO J 1996, 15:1028-l 036. demonstrates the This paper, along with Uhl and Miller, 1996 [lo’], Hls+Asp-+His+Asp phosphorelay of the BvgSlBvgA sensory transduction system. Uhl MA, Miller JF: Central role of the BvgS receiver as a phosphorylated intermediate in a complex two-component phosphorelay. J Biol Chem 1996, 271:33176-33160. This paper, along with Uhl and Miller, 1996 [9-I, demonstrates the His+Asp+His+Asp phosphorelay of the BvgSlBvgA sensory transduction system. 11.
Burbulys D, Trach KA, Hoch JA: Initiation subtilis is controlled by amulticomponent 1991, 64:545-552.
of sporulation phosphorelay
12.
Appleby JL, Parkinson JS, Bourret RB: Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell 1996, 86:845-848.
Wang L, Grau R, Perego M, Hoch JA: A novel histidine kinase inhibitor regulating development in Bacillus subtilis. Genes Dev 1997, 11:2569-2579. A protein inhibitor of KinA, designated Kipl, was discovered. This is the first report of a protein of this functional class. 14.
Perego M, Hoch JA: Protein aspartate phosphatases control the output of two-component signal transduction systems. fiends Genef 1996, 12:97-l 01.
15.
Cotter PA, Miller JF: A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvgregulated antigens. MO/ Microbial 1997, 24:671-685.
16.
TIGR Microbial Database on World Wide Web URL: http://pendant.mips.biochem.mpg.de/frishman/pendant.html http://www.tigr.org
Skorupski K, Taylor RK: Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli MO/ Mkrobiol 1997, 25:1003-l 009.
18.
Miller VL. Mekalanos JJ: Svnthesis of cholera toxin is oositivelv regulateb at the transcriptional level by toxR. froc Nit/ Acad’ Sci USA 1984, 81:3471-3475.
19.
DiRita Vj, Neely M, Taylor RK, Bruss PM: Differential expression of the ToxR regulon in classical and El Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression. PIOC Nat/ Acad Sci USA 1996, 93:7991-7995.
20.
Hase CC, Mekalanos JJ: TcpP is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Nat/ Acad Sci USA 1989, in press.
21.
Kovach ME, Shaffer MD, Peterson KM: A putative integrase defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae. Microbiology 1996, 142:2165-2174.
expression
Cotter
and Miller
25
25.
Reich KA, Schoolnik GK: The light organ symbiont Vibrio fischeri oossesses a homoloa of the Vibrio cholerae transmembrane transcriptional activator ToxR. J Bacterial 1994, 176:3085-3088.
26.
Passador L, Cook JM, Gambello MJ, Rust L. lglewski BH: Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 1993, 260:112711 30.
27.
Pearson JP, Passador L, lglewski BH, Greenberg EP: A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc Nat/ Acad Sci USA 1995, 92:1490-l 494.
28.
Ji G, Beavls RC, Novick RP: Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. froc Nat/ Acad Sci USA 1995, 92:12055-l
2059.
Ji G, Beavis R, Novick RP: Bacterial interference caused by 29. .. autoinducing peptide variants. Science 1997, 276:2027-2030. Demonstration that peptIde pheromones that activate agr-dependent gene expression in one strain can inhibit expression in another. 30.
Pestova EV, Havarstein LS, Morrison DA: Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. MO/ Microbial 1996, 21:653882.
31.
Cauwels A, Charpentier E, Tuomanen E: Multifocal defects in pneumococcal physiology caused by disruption of the ActHActR signal transduction system MO/ Microbial 1997, in press.
32.
Jones BD. N, Ghori S, Falkow: Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer’s patches J Exp Med 1994, 180:15-23.
33. .
Monack D, Rapach B, Hromockyj AE, Falkow S: Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc Nat/ Acad Sci USA 1996, 93:9833-9838. Provides evidence that induction of macrophage apoptosis in response to invasion by Sal/none//a depends on the activation state of the macrophage. 34.
Chen LM, Kaniga K, Galan JE: Salmonella spp are cytotoxic cultured macrophages. MO/ Micro 1996, 21 :l 101-l 115.
35.
Penheiter KL, Mathur N, Giles D, Fahlan T, Jones BD: Noninvasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer’s patches. MO/ Micro 1997, 24:697-709.
36.
Galan JE: Molecular genetic bases of Salmonella host cells. MO/ Microbial 1996, 20:263-271,
and
1 7.
gene
Champion GA, Neely MN, Brennan MA, DiRita VJ: A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains. MO/ Microbial 1997, 23:323-331. Insightful discussion of the evolution of the ToxR regulon.
in B. Cell
13. .
virulence
24. .
9. .
10. .
of bacterial
for
entry into
3% .
Bajaj V, Lucas RL, Hwang C, Lee C: Coordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hi/A expression. MO/ Microbial 1996, 22:703-714. This paper, along with Johnston et al., 1996 [38’], shows the hierarchical regulation of Salmonella invasion genes by regulatory elements that are not encoded within Salmonella pathogenicity island I. 38. .
Johnston C, Pegues DA, Hueck CJ, Lee CA, Miller SI: Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response-regulator superfamily. Mel Microbial 1996, 22:715-727. This paper, along with Baja; ef al, 1996 [37-I, shows the hierarchical regulation of Salmonella invasion genes by regulatory elements that are not encoded within Salmonella pathogenicity island 1. 39.
Groisman EA, Chiao E, Lipps CJ, Heffron F: Salmonells typhimurium phoP virulence gene is a transcriptional regulator. froc Nat/ Acad Sci USA 1989, 86:7077-7081.
40.
Waldor MK, Mekalanos JJ: Lysogenic conversion by a filamentous phage encoding cholera toxin [see comments]. Science 1996, 272:191 O-1 914. Describes the discovery of the filamentous phage, CTX$, encoding cholera toxin.
Miller SI, Kukral AM, Mekalanos JJ: A two-component regulatory system (phoP phoa) controls Salmonella typhimurium virulence. Proc Nat/ Acad Sci USA 1989, 86:5054-5058.
41.
Soncini FC, Groisman EA: Two-component regulatory systems can interact to process multiple environmental signals. J Bacreriol 1996, 178:6796-6801,
23.
42.
Gunn JS, Miller SI: PhoP-PhoQ activates transcription of pmr.46, encoding a two-component regulatory system involved in
gene
22. .
Mel S. Mekalanos JJ: Modulation of horizontal gene transfer in pathogenic bacteria by in viva signals Cell 1996, 87:795-798.
26
Host-microbe
interactions:
bacteria
Salmonella fyphirnurium antimicrobial Bacterial 1996, 178:6857-6864. 43.
peptide resistance.
J
Libby SJ, Goebel W, Ludwig A, Buchmeier N, Bowe F, Fang FC, Guiney DG, Songer JG, Heffron F: A cytolysin encoded by Salmonella is required for survival within macrophages. Proc Nat/ Acad Sci USA 1994, 91:489-493.
44.
Buchmeier N, Bossie S, Chen CY, Fang FC, Guiney DG, Libby SJ: SlyA, a transcriptional regulator of Salmonella typhimurium, is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages. infect lmmun 1997, 65:3725-3730.
45.
Lindgren SW, Stojiljkovic I, Heffron F: Macrophage killing is an essential virulence mechanism of Salmonella typhimorium. Proc iVat/ Acad Sci USA 1996, 93:4197-4201.
46.
Chen CY, Eckmann L, Libby SJ, Fang FC, Okamoto S, Kagnoff MF, Fierer J, Guiney DG: Expression of Salmonella fyphimurium rpoS and rpob-dependent genes in the intracellular environment of eukaryotic cells. infect lmmun 1996, 64:47394743.
47.
Mahan MJ, Slauch JM, Mekalanos JJ:Selection of bacterial virulence genes that are specifically induced in host tissues [see comments]. Science 1993, 259:686-688.
48.
Heithoff DM, Conner CP, Hanna PC, Julio SM, Hentschel U, Mahan MJ: Bacterial infection as assessed by in viva gene expression. Proc Nat/ Acad Sci USA 1997, 94:934-939.
49.
Wang J. Mushegian A, Lory S, Jin S:Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in viva selection froc Nat/ Acad SC; USA 1996, 93:10434-l 0439.
50.
Young GM, Miller VL: Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis. MO/ Microblol 1997, 25:319-328.
51.
Camilli A, Beattie DT, Mekalanos JJ: Use of genetic recombination as a reporter of gene expression. Proc Nat/ Acad Sci USA 1994, 91:2634-2638.
52.
Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW: Simultaneous identification of bacterial virulence genes by negative selection. Science 1995, 269:400-403.
53.
Hensel M, Shea JE, BHumler AJ, Gleeson C, Blattner F, Holden DW: Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia co/i K-l 2. J Bactenol 1997, 179:l 105-l 111.
54. .
Shea JE, Hensel M, Gleeson C, Holden DW: Identification of a virulence locus encoding a second type Ill secretion system in Salmonella typhimurium. Proc Nat/ Acad Sci USA 1996, 93:2593-2597. Describes the discovery of a second Salmonella pathogenicity island, SPIZ, encoding a Type Ill secretion system. 55.
Hensel M, Shea JE, Raupach B, Monack D, Falkow S, Gleeson C, Kubo T, Holden DW: Functional analysis of ssal and the ssaK/U operon. 13 genes encoding components of the type Ill secretion apparatus of Salmonefla pathogenicity island 2. MO/ Microb,o/ 1997, 24:155-l 67.
56.
Valdivia RH, Falkow S: Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. MO/ Microhiol 1996, 22:367-378.
57. ..
Valdivia RH, Falkow S: Fluorescence-based isolation of bacterial genes expressed within host cells. Science 1997, 277:20072011. The power and versatility of differential fluorescent induction (DFI) were demonstrated by these authors. DFI was used to select Salmonella promoters with enhanced expression in macrophages compared within medium alone; expression of these promoters within host cell tissues in viva was then monitored via green fluorescent protein technology.
of
regulation
In vivo and ex vivo expression
aacterlal
r--_--_,-l
vwlence
gene
__~-__I_---
_____
_
Peggy A Cotter* and Jeff F Millet-t Bacteria
are remarkably
to survive
adaptable
and multiply
environments. of genetic
Adaptability
information
mechanisms
conferring
particular
situation
deleterious
organisms
expression. while bacteria
within
host cells and tissues
Being
of little or no use to the bacterium
specific factors
stages
to appreciate bacteria
gene
the complexities
to this rule.
except
during
to tight and coordinate advances,
we are beginning
of the interactions
and their hosts. The ability to probe
regulation
gene
in viva has broadened
between
virulence
our perspectives
on
pathogenesis.
Addresses Department of Microbiology and Immunology, University of California at Los Angeles School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-l 747, USA *e-mail: [email protected] +e-mail: [email protected] Current
Opinion
in Microbiology
1998,
1 :17-26
http://biomednet.com/elecref/1369527400100017 0 Current Biology Ltd ISSN
1369-5274
Abbreviations CSP CT
competence stimulating peptide cholera toxin
DFI
differential fluorescence
FACS FAE GFP
fluorescence activated cell sorting follicle associated epithelium green fluorescent protein histidine phosphotransfer domain homoserine lactone in viva expression technology
HPt HSL IVET SPI
Salmonela
pathogenicity
STM
signature tagged
TCP
toxin co-regulated
hosts [Z]. Diffusible signals can the bacteria themselves, conferring cell density [3,4]. Finally, sensed by direct physical
also be produced by the ability to measure
host cells can apparently contact between bacteria
be and
target eukaryotic cells [So*]. Signals that affect virulence control systems in vitro can be used to effectively identify regulated genes; however, in many cases their relationship to signals important in viva is not well understood.
on and
cycle, these accessory
subject
As a result of recent
or
of virulence to multiply
gene
in a
unnecessary
are no exception
of the infectious
are nearly always
regulation.
advantage
are not. Expression
that allow pathogenic
and by the
In general,
or survival
are expressed,
functions
hostile
by the complement
to an organism
gene
a growth
that are able
and sometimes
is determined
available
that control
products
products
in diverse
induction
island
mutagenesis pilus
Introduction Our understanding of the mechanisms that govern bacterial virulence gene expression is relatively advanced compared with our understanding of when and why virulence gene regulation actually occurs. A diverse array of extracellular signals have been identified that modulate virulence gene expression in vitro. Many of these signals are general and seemingly nonspecific physical or chemical cues [ 1’1. In other cases bacteria have evolved to recognize specific metabolic products produced by their respective
Recognition that virulence loci are tightly and precisely regulated has spurred a flurry of activity towards identifying genes that are specifically expressed i?i viva. While this is an important goal, the ability to detect genes that are truly ‘in viva induced’ requires a detailed understanding of the relevant ex vlvo conditions. This in turn necessitates an appreciation of the entire infectious cycle and the array of environments that characterize it. Pathogenesis can be viewed as a developmental process characterized by a progression of spatially and temporally defined events. The most pertinent questions to ask regarding virulence gene control involve relative gene expression in relevant microenvironments. Virulence gene regulation is therefore best considered in a truly ecological context, and it is reasonable to expect that ecological complexity will be mirrored by the complexity of the regulatory circuitry that modulates gene expression. Our aim in this review is to focus on a selected set of virulence regulatory systems that illustrate general principles. Useful experimental approaches and results obtained in viva will be emphasized. Although we focus exclusively on pathogens of mammals, many of the same principles are apparent from studies of bacterial-plant interactions [6] and symbiotic
Virulence Virulence
interactions
with
invertebrate
hosts
[7].
regulatory components expression
is usually
governed
by
signaling
and regulatory mechanisms similar to those that control genes that are not specific to pathogenesis. One of the most regulatory
commonly encountered families of virulence proteins is the two-component group of signal
transducers. These couple a diverse array of signals to multiple effector outputs using regulated phosphotransfer reactions. Hallmarks of two-component systems are their modularity and accompanying adaptability. These features are highlighted by several recent developments. Three types of conserved signal transducing components are found in two-component systems. These are illustrated in Figure la using the Salmonella PhoQ/PhoP and Bordetella BvgS/BvgA virulence control systems as specific examples. In both cases signal transduction begins with autocatalyzed phosphorylation of a sensor kinase in the
18
Host-microbe
interactions: bacteria
presence of activating environmental conditions and ATP. This reaction is catalyzed by a transmitter domain which is linked to an input module with direct or indirect sensory capabilities. The input domains of PhoQ, BvgS and most other sensor proteins span the cytoplasmic membrane and they are generally assumed to control the autophosphorylation reaction. In vitro, PhoQ activity can be modulated by hlgz+ and CaZ+ [8] while BvgS responds to temperature, SOqz- and nicotinic acid [9’]. Sensor proteins serve as substrates for phosphocransfer reactions between transmitter phosphohistidines and conserved aspartic acid residues in receiver domains. For PhoQ/PhoP, this activates the output domain of the PhoP response regulator, allowing activation and repression of target gene transcription. In contrast, the BvgS/BvgA system operates by a more complex phosphorelay [9*,10’]. This signaling mechanism was initially discovered by Burbulys et al. [ll] in their elegant studies of Bacillus subtilis sporulation. As shown for BvgS, two receiver domains and a histidine phosphotransfer domain (Hpt) are involved in the phosphorelay. Phosphorylation of the BvgS receiver results in phosphoryl group transfer to water, an abortive event, or transfer to a conserved histidine in the Hpt. The phosphorylated Hpt is the substrate for phosphotransfer to the cognate receiver in the BvgA response regulator. This type of phosphorelay is widely distributed in nature [12]. The individual components can be connected in a variety of configurations or be contained on completely separate proteins. Why might a complex phosphorelay exist? PhoQ/PhoP and BvgS/BvgA perform apparently similar functions, but in the latter case twice as many phosphotransfer reactions are required. For any integrated circuit, the exact relationships between input and output will depend on the specifications of the components. hlultiple components with different properties could therefore fine-tune this relationship. An alternative but not mutually exclusive idea is that multiple steps allow multiple points for control. studies
This has received recent support, of sporulation. In B. subtih, proteins
again from that inhibit
autophosphorylation [13’] as well as receiver-specific phosphatases [14] have recently been discovered. It is also important to consider that two-component systems may not simply function as on/off switches for gene expression. This is especially apparent in the case of BvgAS [15]. In B. bromhiseptica, a respiratory pathogen of many fourlegged mammals, this system activates genes required for respiratory tract colonization and represses genes involved in motility. A class of BvgAS-regulated polypeptides expressed between these two extremes has recently been systems can discovered (Figure lb) [15]. T wo-component therefore control multiple waves of gene expression. An examination of available genomic sequences indicates that the number of two component family members present in a bacterium can vary dramatically [16]. For example, Helicobacterpylori appears to encode four sensors and six response regulators; Hemophihs ir@~enzae encodes four
sensors and coli encodes
seven response an estimated
regulators; 16 sensors
while Escherkhia and 40 response
regulators! It is intriguing to speculate that the number of two component family members encoded in a particular genome may correlate with the ecological complexity of the bacterium’s life cycle.
Hierarchical control and evolution of the Vibrio cholefae virulence regulon V&o dolerae is a motile, Gram-negative, curved rod which is endemic to ocean waters where it is either free-living or exists in association with copepods and other crustacea. When ingested by humans, this noninvasive pathogen causes severe secretory diarrhea. Adherence of bacteria to the intestinal epithelium is mediated in part by the toxin co-regulated pilus (TCP), and massive fluid loss results from the action of a potent ADP-ribosylating exotoxin (cholera toxin, CT). CT and TCP expression has been known for many years to respond to temperature, pH and other environmental conditions [17]. Early studies by Miller and hlekalanos [18] identified the toxRto.vS regulatory locus, which controls CT and TCP expression as well as the expression of more than 20 additional genes. Subsequent genetic and biochemical analyses contributed to the elucidation of the regulatory hierarchy shown in Figure 2 [17]. ToxR/ToxS control of virulence gene expression is mediated largely via ToxT, and it appears that differential expression of CT and TCP in the V cholerae classical and El Tor biotypes stems from differences in ToxR/ToxS-mediated induction of toxT expression [ 191 which may actually occur via TcpP/TcpH [ZO]. Genes within the ToxT-dependent pathway encode accessory colonization factors (a@, additional regulatory loci (tcpZ, tcpP and tcpH), cholera toxin &A and ctxB), TCP (tcpA-F) and toxT itself. Other genes, such as ompU and ompT, appear to be directly regulated by ToxR/ToxS. This hierarchically organized regulatory circuit may reflect a need to integrate various signal inputs to maintain separate but coordinated control of multiple genes. Two recent observations provide insights regarding the evolution of the ToxR regulon. The first is the recognition by Kovach et a/. [Zl] that the linked tcp, toxT and ad loci reside on a large pathogenicity island. The second is the discovery by Waldor and hlekalanos [22*] that OXA and ctxB are encoded on a lysogenic filamentous bacteriophage designated CTX@. These observations suggest that the hierarchical structure of the ToxR regulon reflects the horizontal acquisition of genetic elements encoding determinants for pathogenesis in humans. It has been proposed that ToxR/ToxS originally evolved to control outer membrane protein synthesis in an ancestor lacking the tcp-acf pathogenicity island and CTX$ [23,24’]. In support of this hypothesis, ToxR/ToxS homologues are present in nontoxigenic, non-TCP-expressing 1,: dolerae strains [l&21]. ToxR/ToxS homologues are also present in Vibriofischeri and Photobactetiwn sp. strain SS9, marine microbes that are not associated with human disease
In viva and ex viva regulation of bacterial virulence gene expression
Cotter
and Miller
19
Figure 1
(a)
Environmental signals Transmitter
Input
Receiver
PhoQ
Environmental signals
Output
PhoP
4
Transmitter
Input
Receiver
Hpt
Receiver
BvgS
Output
BvgA
oBvg+
Bvgi
Bvg-
?
Flagella urease
2.
3.
A:‘,$,“;s 1.
Current Opinion on Microbiology The two-component
group
of signal transducers
regulate
(a) The Salmonella
gene expression.
transduction systems. histidine residue (Hl)
The input domains of PhoQ and BvgS sense environmental in the transmitter domain. The transmitter phosphohistidines
aspartic
(Dl)
acid residues
regulation
of target
in the receiver
gene transcription.
domains.
For Phoa/PhoP
The phosphorylation
phosphorylation
of the BvgS
Dl
present
2). and Bvgmodulation
in the input domains.
phase
by environmental
by the expression
(b) Relative expression
loci (line 3) are indicated
of adhesins
conditions. and toxins,
on the vertical
ln viva experiments is necessary
expression of motility and other co-regulated Bvgi phase expressed antigens is unknown.
phenotypes,
of Dl
receiver
histidine (H2) on the histidine phosphotransfer domain (Hpt). The phosphotylated conserved aspartic acid residue (D2) in the BvgA receiver domain; this activates sequences
[24*,25]. The fact that TCP provides the receptor for CTXQ, and that both the tcp-acf cluster and ctxAB are controlled by ToxT, suggests their co-evolution may have preceded their association with 1! cholerae. Once introduced into II dolerae, control of these genes may
in transfer
the output
domain
of a phosphoryl
loci (line l), Bvg intermediate
axis indicates
for respiratory
is advantageous
and Bordetella
BvgS/BvgA
sensory
of PhoP resulting group
in
to a conserved
Hpt serves as a substrate for phosphotransfer to the the BvgA output domain. TM corresponds to transmembrane
with B. bronchiseptica
and sufficient
activates
results
levels of Bvg+ phase
axis. The horizontal
PhoP/PhoQ
signals which result in autocatalyzed phosphorylation of a serve as substrates for phosphotransfer to the conserved
phase-locked tract
for growth
phase
the phase of the organism
infection,
mutants
indicate
while the Bvg-
under nutrient-limiting
loci (Bvgi) (line
which
results
the Bvg+ phase, phase,
conditions
from
characterized
characterized
by the
(151. The function(s)
of
have come under ToxR/ToxS control via ToxT, thus converting ToxR/ToxS into a regulator of virulence gene expression. Interestingly, since TCP serves as the receptor for CTX$, ToxR/ToxS has been converted into a regulator of horizontal gene transfer as well.
20
Host-microbe
interactions: bacteria
Figure 2
B ToxR
Outer
membrane
ToxS
inner membrane
TCP-ACF
The K cholerae ToxR/ToxS regulon. ToxR and ToxS are cytoplasmic membrane expression of ompT. ToxR/ToxS controls expression of the cholera toxin genes
proteins.
ToxR/ToxS activates expression of ompU and represses encoded on the bacteriophage CTXQ and
(ctuA and CM)
activates the expression of genes on the TCP-ACF pathogenicity island, including genes encoding the toxin co-regulated pilus (tcp genes). ToxR/ToxS may control expression of accessory colonization factors (acf genes) indirectly via ToxT. It appears that TcpP/TcpH, which are also predicted to be cytoplasmic membrane proteins, activates expression of toxT, and ToxT activates tcp, acf, and ctu loci [20,211. For simplicity, regulatory
functions
of fcp loci are not shown.
Recognition that the primary virulence determinants of V cholerae are contained on discrete genetic elements suggests that horizontal gene transfer provides a mechanism for converting a nonpathogenic marine microbe into a pathogen responsible for severe human disease. It is also interesting to speculate on the function of the virulence regulatory system throughout the infectious cycle. ToxR/ToxS-activated genes are certainly required in viva, but they may also provide unrecognized advantages within the marine environment. A complete analysis of virulence gene expression by this important bacterium will require model systems that reflect the ecological niches characterizing
the entire
infectious
cycle.
Quorum sensing Quorum sensing differs from other signal-dependent responses in two major respects: the signal is produced by the bacteria themselves and the regulatory system measures bacterial cell density and growth phase. Several Gram-negative bacteria produce N-acyl homoserine lactone (HSL) molecules which serve as membrane-permeable signals. HSLs accumulate during growth and activate gene expression by binding to cytoplasmic receptor proteins that function as HSL-dependent activators [3]. Transcriptional activation is highly dependent on HSL concentration. This type of quorum sensing is found in a
variety of Gram-negative bacteria that establish symbiotic or pathogenic associations with plants or animals. In the opportunistic human pathogen Pseudomonas aeruginosa, two quorum-sensing systems (/as and I-I?/) regulate virulence gene expression [26,27]. Quorum sensing in Gram-positive bacteria follows a distinctly different paradigm [4]. The signaling molecule is usually a post-translationally processed, secreted peptide pheromone, which is sensed by the input domain of a two-component sensor. For Staphylococcus aureus, it has recently become apparent that multiple virulence factors are coordinately regulated by the quorum-sensing system summarized in Figure 3 [28]. Most of the signaling components are encoded by the agrBDCA operon. The agrD product is processed to yield a 7-9 amino acid peptide which is secreted into the culture supernatant. Pheromone production is dependent on AgrB, but the exact role of this protein is unclear. During post-exponential phase growth in vitro, the concentration of pheromone reaches a critical threshold level sufficient for activating AgrC, a transmitter-containing sensor protein with multiple transmembrane sequences. Phosphorylation of the AgrA response regulator apparently leads to transcriptional activation of two divergent promoters. The P2 promoter transcribes the agr operon itself, thereby amplifying the
In viva and ex viva regulation of bacterial virulence gene expression Cotter and Miller
sensory
response.
The
P3 promoter
initiates
expression
of a 0.5 kb transcript, RNAIII, which is the actual effector molecule. Although its mechanism of action is not yet clear, RNA111 appears to act primarily at the level of transcription of regulated genes. Genes encoding protein A, fibronectin-binding protein, coagulase and other surface proteins with potential roles in establishing infection and evading host defenses are preferentially expressed during exponential growth if1 vitro. This is before agr becomes active, and activated agr represses their transcription. Conversely, genes encoding a variety of excreted factors are induced when agr is active. These include alpha-hemolysin, enterotoxin B.
toxic shock
syndrome
toxin,
and
In a recent report by Ji et al. [29”], different strains of S. aureus were shown to produce structurally different peptide pheromones. Pheromones released by one strain could repress virulence gene expression in another. This ‘bacterial interference’ (so called because of the production of inhibitory peptides) could potentially exclude competing strains from occupying sites for colonization. The observation that exogenous peptides can inhibit staphylococcal virulence may lead to new therapeutic modalities for this and other Gram-positive pathogens. sensing is also used by Streptococcus regulate transformation competence and other phenotypes as well. Two separate two-component systems are involved in controlling transformation. The ComD/ComE proteins sense and respond to the competence-stimulating peptide (CSP) [30], and the ActR/ActH system is required for CSP production [31]. Mutation of actH also results in impaired nasopharyngeal colonization in rats, making ActR/ActH the first two-component system shown to be important for pneumococcal virulence [31]. Peptide-dependent
pneumoniae
to
What, exactly, is the linking virulence gene population or growth
selective advantage conferred by expression to the size of a bacterial phase? In the case of S. aureus,
the switch between the expression of surface proteins and excreted toxins may reflect a dichotomy between colonization and disease. Do greater numbers of bacteria provide an opportunity for host invasion? Does entry into post-exponential phase indicate impending bacterial doom? Perhaps the most important question is how to address the relevance of quorum sensing experimentally. It is clear that the agr regulon is needed for S. aureus virulence, indicating the importance of inducing exoprotein production. Less apparent is the need for repressing the expression of surface proteins during postexponential growth, and the reason why excreted factors are repressed in the exponential phase. An approach that might yield insights is to ‘rewire’ the control system to abrogate density-dependent regulation. Animal models that measure colonization, as compared with only disease, will be also required to understand the relationships between
cell density-
and growth-phase-dependent
regulation
21
and
pathogenesis.
The Salmonella paradigm: probing virulence gene expression in vi110 Sa/monel/a &Ohimutium causes
self-limiting gastroenteritis in humans. Infection of mice, however, results in a systemic illness similar to human typhoid fever. Following ingestion, S. typhutium 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 [32]. Destruction of the M cells quickly follows, forming a gap in the FAE which allows invasion of adjacent enterocytes. This damage also allows S. ~phmurium to gain access to the reticuloendothelial Invasion of activated macrophages appears to system. induce their apoptosis [33*,34] while invasion of resting macrophages results in altered membrane trafficking and prevention of phagosome-lysosome fusion. Survival of S. t)phimutium in nonactivated macrophages facilitates bacterial spread to the spleen, liver and bone marrow. The organism’s ability to survive in these various intracellular and extracellular environments requires a vast array of gene products. These are encoded on at least two pathogenicity islands, a virulence plasmid, and various linked and unlinked chromosomal loci. As seen with li chokrae, horizontally acquired genetic elements have apparently co-opted chromosomal control factors, resulting in a hierarchically organized complex regulatory network. Virulence genes in S. qphimurium appear to be controlled primarily in response to whether the bacterium is in an intracellular or extracellular compartment (Figure 4). Two regulatory systems of major importance are SirA/HilA and PhoP/PhoQ. Extracellular S. &Gimutium appear to be primed for invasion of a variety of host cell types. Invasion of M cells in viva and epithelial cells and macrophages in vitro requires a Type III secretion system encoded on the SPl Salmonella pathogenicity island located at centisome 63 [1*,35,36]. This Type III system triggers a complex cascade of signaling events in the host cell that results in marked cytoskeletal rearrangements, membrane ruffling and bacterial uptake by macropinocytosis. In Yersinia species, contact with host cells via a similar Type III secretion system results in the delivery of antiphagocytic proteins directly into the target cell cytoplasm rather than in internalization of the bacterium. By including a transcriptional repressor in the class of exported proteins,
lhinia contact
couple virulence gene [5**]. In S. Ilp/rimurium
regulation with genes encoding
host cell the SPIl
Type III system, and its secreted targets, are positively activated by HilA [37*]. Expression of hi/A was recently shown to require sirA, which encodes a two-component response regulator [38*], suggesting the existence of a cognate sensor protein that responds to cues in the extracellular milieu. Since the original sirA mutant could also be complemented by the unlinked loci sirB and sid, control of MA by sirA may not be direct.
22
Host-microbe
interactions: bacteria
Figure 3
S. aureus strains Interference
U
AgrB
P3
(?)
P2
agrA
agrC
a
AgrB
(?) a
RNAM R n Peptide 0 Protein
A
coagulase FBP
Capsule TSST a-hemolysin exotoxin
B Current Opmon in Microbiology
Quorum
sensing
in S. aureus [28,29”1.
of rnaiii and the agrBDCA pheromones are secreted
The agrSDCA
locus encodes
loci in response to the presence by ATP-binding cassette exporter
the AgrC/AgrA
either directly or indirectly, activates expression of capsule, toxic shock syndrome expression of protein A, coagulase and fibronectin-binding protein (FBP). Peptide producing
them can in some cases
inhibit
agr in other strains
two-component
regulatory
system
of peptide pheromones. In many cases, post-translationally proteins; however, such a factor has not been described
and cause
Following internalization by target cells, the PhoP/PhoQ sensory system becomes activated, possibly by recognizing differences in Caz+ and/or Mg2+ ion concentrations between extracellular and intracellular environments [S]. Activated PhoP simultaneously represses prgs (PhoP/PhoQrepressed genes), including those encoding the SPIl Type III secretion system, and activates expression of pags (PhoP/PhoQ-activated genes). The ability to survive in macrophages is crucial to Sa/mone//a pathogenesis and the requirement for expression of pag loci is well established in this regard (39,401. It was recently shown
expression
toxin (TSST), a-hemolysin and exotoxin B, and represses pheromones which activate the agr regulon in strains
‘interference’
that
that controls
processed peptide for the agr system. RNAIII,
[29”1.
macrophage survival results, at least in part, from and pmd expression. These are pags that encode yet another two-component regulatory system. PmrA/PmrB control resistance to cationic antimicrobial peptides such as those present in macrophage phagosomes [41,42]. Other intracellularly active regulatory factors include SlyA, which contributes to survival in macrophages and destruction of M cells [43,44], EnvZ/OmpR, which contributes to macrophage killing [45], and RpoS, which contributes to destruction of hl cells and spv-mediated growth in the liver and spleen [46]. Virulence genes expressed by intracellu-
pmfA
In viva and ex viva regulation of bacterial virulence gene expression Cotter
and Miller
23
Figure 4
I
Extracellularly
Invasion
of
I
located salmonella
orgA
prgKJIH
sipADCB
MA iagf
invCBAEGf
spa TSROPONM
invH @ I,
----------------
T
(E) [FEiF)&-s~Ra”,_
0
(TiE$gj [killing
j A
_fJ
P
S”
P
I
_J
-\
II
ssaUTSROPONVMLKJ
Y
V
PhoQ
EnvZ
SP12
(polymyxin6’)
OM
lntracellularly
located salmonella
Current Opmon
Virulence
gene regulation
in S. typhimurium
[35,36,37’,36’1.
The SirA/HilA
regulon,
which
is expressed
primarily
when
Salmonella
I” Mwobiology
are
extracellularly located (i.e. not found in host cells), is shown above the dotted line. Expression of hi/A requires SirA, a possible two-component response regulator. HilA positively activates the spa, inv and prg operons of the Salmone//a pathogenic&y island 1 (SPIl) which encode proteins that form the Type Ill secretion apparatus dotted genes
apparatus
and allow the invasion
(TIII). The Sip proteins,
of epithelial
whose
cells by S. typhimurium
expression
is activated
and the invasion
by Inf, are exported
and induction
of apoptosis
through
the Type Ill secretion
in macrophages.
Below
the
line, genes expressed primarily by intracellularly localized Salmonella are shown. PhoP/PhoQ represses expression of SPll -encoded (bar leading up to dotted line) and activates expression of genes and operons (i.e. s/yA and pag loci) required for intracellular survival.
Other regulons contributing to the phenotypes of intracellularly located S. fyphimurium are also shown. The function system encoded on SP12 is not known. The bacterial inner membrane (IM) and outer membrane (OM) are shown.
larly localized Salmonella, therefore, array of regulatory systems. In vivo expression
are controlled
by an
technology
Genetic tractability and the availability of 171 vitro and irr viva models for invasion and pathogenesis have made Salmonella an excellent system for developing methods to identify virulence genes and monitor their regulation. Ztr viva expression technology (WET) is a promoter trap scheme in which random DNA fragments are cloned in front of promoterless purA and /acZ genes such
of the Type Ill secretion
that genes that are expressed in vivo but not under specfic conditions if/ vitro can be identified (47). The assumption is that at least some virulence genes will conform to this overall pattern of expression. The IVET concept was a breakthrough and it has been successfully used with Salmonella, Pseudomonas, and Ye&zia species [48-SO]. The initial IVET system required sustained gene expression in animals. A recently developed version based on site-specific recombination in response to resolvase (tnpR) gene expression allows the identification of loci that are only transiently expressed i7z viva [51]. A potential
24
Host-microbe
disadvantage
requires j?~ vivo
interactions: bacteria
to the
IVET
approach
the proper choice induced promoters
is that
its success
of ex viva conditions, will only be detected
as if
the conditions chosen for growth in culture efficiently repress their expression. The original methodology was developed using mice versus agar as growth media. IVET’s maximum potential, however, may lie in identifying genes that are differentially expressed between two or more environments which are truly relevant to a pathogen’s infectious
cycle.
Signature-tagged
system [53,54*]. Although SPIZ-encoded genes appear not to be involved in invasion, survival or replication in macrophages, or induction of apoptosis, SPIZ mutants are severely attenuated after oral or intraperitoneal inoculation of mice [52,54’,55]. STM will undoubtedly lead to the discovery of previously unrecognized virulence genes in many bacterial pathogens. Green fluorescent protein technology The ultimate goal in understanding bacterial virulence gene regulation is to determine the dynamics of gene expression throughout the infectious cycle. Differential fluorescence induction (DFI) uses fluorescence-activated sorting (FACS) basis of their
to screen ability to
animals. It was shown that all of the promoters identified using macrophages it) vitro were expressed in splenocytes and hepatocytes following intraperitoneal inoculation of mice [57”]. This system represents a powerful new technology for identifying genes that are expressed in response to specific environments and for monitoring expression patterns during the process of pathogenesis.
Conclusions
mutagenesis
A more recent technology, termed signature-tagged mutagenesis (STM), is designed to identify genes that are required for growth or survival 171 v&o, but not for growth irr vitro [SZ]. SThl differs from IVET in that assumptions regarding gene expression are not required. STM provides a mechanism for identifying individual avirulent mutants in large pools of mutagenized bacteria, allowing a relatively small number of animals to be used for the initial screen. STM is a transposon-based mutagenesis scheme in which each transposon is marked with a different DNA sequence tag, permitting the comparison of mutants that survive passage through an animal with those that survive passage through culture medium. This powerful screening method led to the discovery of a second pathogenicity island in Salmonella, SPIZ, which appears to encode a Type III secretion
cell the
and at least one gene encoded on SPIZ were also detected in this screen. Because identified promoters are fused to gfp, expression can be monitored in tissues of infected
for active promoters drive expression of
on the
Aequorea Victoria jellyfish green fluorescent protein (GFP) [56]. DFI facilitates the selection of promoters that are differentially expressed in response to defined environmental conditions, including intracellular localization in eukaryotic cells. Major advantages of this approach include sensitivity and adaptability. Gene regulation can be observed at the level of individual bacterial cells, and variability in expression levels within a population can be determined. Bacteria, or eukaryotic cells containing them, can also be sorted on the basis of fluorescence intensity. Using DFI, Valdivia and Falkow [57”] identified several Salmonella promoters that were well expressed in macrophages yet poorly expressed in tissue culture medium alone [57”]. Nine of the 14 loci identified were regulated by PhoP/PhoQ. An EnvZ/OmpR-regulated gene
hluch of this discussion has focused on the adaptability of bacterial pathogens. The examples described above highlight another feature of these organismsnamely their remarkable diversity. Although similar components are often used to couple signals to responses, they are adapted by different bacteria to accomplish distinctly different tasks. An excellent example is the signaling modules that constitute two-component systems. They are used by S. aureus to sense other bacteria, by Salmonella species to differentiate extracellular from intracellular environments, and by B. bronchiseptica to sense whether they are inside a host or not. Type III systems are another example of similar components being used for significantly different reasons. Diversity can also be seen in the organization of virulence control networks. These regulatory circuits appear to be quite dynamic in an evolutionary sense, having the ability to adapt existing regulatory systems to control horizontally acquired genes. Over the last few years, our ability to probe virulence gene regulation has advanced considerably. Combining new methodologies with appropriate in vivo and ex viva models will highlight the diverse mechanisms pathogens use to adapt to their mammalian hosts.
Acknowledgements W
thank refcrcnced and unrcfcrenced cnllcagues for a wealth nf inrerexing expcrimenrs, data and insrghts, and hlingshun Liu for help with technical aspects of the review. The authors are supported by grants from the National Insrirute nf Health (grants A138417 and AI38955) and the American Cancer Socict~ (grant Ihl-791).
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