Lecture 4 Signal Perception Flashcards
What signals do bacteria respond to?
ENVIRONMENTAL CUES INCLUDE: Temperature Acidic pH Ions – e.g. Mg2+, Fe3+ Population density (QS) Osmolarity Presence of antimicrobials Oxygen availability Nutrient availability
QS of Gram-positives – a reminder…
‘Auto-inducing peptides’ (AIP) instead of AHLs
Produced as propeptide before processing and active secretion
AIP does not re-enter cell, but triggers signalling via two-component signalling pathway
Two-component systems (TCSs) – an overview
Characteristically, TCSs include two different proteins – a sensor kinase protein located within the cytoplasmic membrane and a partner (“cognate”) response regulator protein present in the cytoplasm.
Sensor kinases detect a signal from the environment & autophosphorylate themselves (using ATP) at a specific histidine residue within the cytoplasmic domain. Consequently, these sensor kinases are sometimes also referred to as histidine kinases. This phosphoryl group is then transferred to the response regulator protein – on to a conserved aspartate residue within the response regulator.
Sometimes, intermediate proteins facilitate a phosphorelay between the sensor kinase and the response regulator. In other cases, it is direct transfer between the two proteins.
In most cases, the response regulator is a DNA-binding protein that regulate transcription. Its phosphorylation results in a conformational change that typically alters DNA-binding affinity. Binding to the relevant promoter region can enhance or inhibit transcription (depending on the specific system).
Phosphatase activity resets the system (removing the phosphoryl group from the sensor kinase and/or the response regulator). In many cases, this phosphatase activity is conferred by the proteins of the TCS themselves.
Common features of TCSs
In many cases, TCSs auto-regulate themselves (comparable to QS)
i.e. the phosphorylated RR enhances expression of the genes encoding the TCS itself
Despite multiple TCSs being present within individual bacterial cells, there exists remarkable specificity between the cognate pairs
i.e. sensor kinases only phosphorylate their specific response regulator
Same genes can be activated by multiple TCSs
This allows the same set of genes to be activated by multiple environmental stressors
Often, we do not know what the activating environmental signals are in vivo!
The EnvZ/OmpR TCS
Outer membrane of Gram-negative bacteria contains channels made of porin proteins; allow transport of small molecules
In Escherichia coli, the most important porins are OmpF & OmpC
The balance between OmpF & OmpC is critical, and corresponds with the osmolarity of the environment
OmpC pores are slightly smaller; favoured during growth at high osmotic pressures
Larger OmpF pores are favoured in more dilute environments, allowing solutes to diffuse into the cell more readily
EnvZ sensor kinase senses osmolarity
EnvZ sensor kinase spans the membrane; periplasmic sensing domain senses the osmolarity of the environment
At low osmolarity, EnvZ is inactive
In response to increasing osmolarity, EnvZ autophosphorylates
OmpR response regulator controls OmpF/C
OmpR is the cognate response regulator for EnvZ; N-terminal domain is the receiver domain, C-terminal domain the DNA-binding domain
Under high osmolarity, transfer of the phosphoryl group from EnvZ to the receiver domain of OmpR enables DNA-binding by OmpR
OmpR activates ompC expression whilst repressing ompF expression
Salmonella species
Gram-negative, facultatively anaerobic bacilli
Normal habitat is animal intestine
Salmonella enterica divided into serotypes
Salmonella enterica serotype Typhimurium (Salmonella typhimurium)
Salmonella enterica serotype Enteritidis (Salmonella enteritidis)
Salmonella enterica serotype Typhi (Salmonella typhi)
Overview of Salmonella infection
Orally-ingested Salmonellae survive the acidic pH of the stomach and preferentially enter M-cells. These M-cells transport the salmonella to the lymphoid cells in the underlying Peyer’s patches – lymphoid tissue that essentially performs immune surveillance for the gastrointestinal system. Salmonella serotypes that are associated with systemic infection will enter intestinal macrophages and will be disseminated throughout the reticuloendothelial system. In contrast, non-typhoidal Salmonella induce a localized inflammatory response, resulting in the influx of PMNs to the intestinal lumen and diarrhoea.
Defences in the GI tract – low pH & bile salts
Acidic pH
Gastric acidity is a 1st line of defence against oral infection route
pH can be as low as 1.5
Bacterial survival requires an acid tolerance response that promotes survival & growth at low pH
Bile salts
Component of bile, which is discharged into small intestine to aid digestion
Antimicrobial activity linked to detergent-like properties
Bacterial resistance is based around decreasing porin expression and boosting efflux systems
Phagocytosis – protection against infection
Macrophages, neutrophils & dendritic cells
Killing of phagocytosed microbes occurs within specialized compartments, termed phagolysosomes
Bactericidal mechanisms in phagolysosome include reactive oxygen species, antimicrobial peptides, and acidification
Bacteria are ingested into the phagosome. The phagosome then undergoes a process of maturation, in which a variety of vesicles including lysosomes fuse with it, delivering components that help kill the ingested bacterial cell. The mature phagosome is called the phagolysosome. They are generally very effective at killing ingested bacteria, but some bacteria have sophisticated mechanisms to evade killing. These bacteria are sometimes referred to as “professional intracellular pathogens”.
Salmonella PhoP-PhoQ & PmrA-PmrB TCSs
PhoP-PhoQ and PmrA-PmrB TCSs are very well characterized. Within Salmonella, they have a distinctive feature that they are connected by the actions of PmrD – a protein which is activated by the PhoPQ system that then protects the phosphorylated PmrA protein from phosphatase activity of PmrB, thus promoting PmrA activation. This means that the same set of genes can be activated by a larger set of stimuli.
PhoPQ contributes to resistance to bile salts
Consequently, activation of the PhoP-PhoQ TCS in the acidic environment of the GI tract can promote resistance to bile
Data from the study by van Velkinburgh et al., 2009 shows that the Salmonella phoP mutants show reduced levels of resistance to bile compared to the wildtype.
PhoPQ & PmrAB; resistance to antimicrobial peptides
Antimicrobial peptides (APs) are positively-charged, and have high affinity for the negatively-charged bacterial cell envelope This interaction with the bacterial membrane is largely responsible for the lethal effect of APs
The PhoPQ AND PmrAB TCSs both control modifications to the lipid A region of the lipopolysaccharide that confer resistance to APs
Most notably, the addition of aminoarabinose (Ara4N) to a negatively-charged phosphate group in the lipid A reduces the net negative charge, thus lowering the affinity for APs
Bacterial resistance to oxidative killing
Superoxide dismutase (SOD) enzymes detoxify superoxide (O2-)
Superoxide dismutase
Cu2+−SOD + O2−»_space;> Cu+−SOD + O2
Cu+−SOD + O2− + 2H+»_space;> Cu2+−SOD + H2O2
Catalase / peroxidase
2H2O2»_space;> 2H2O + O2
The superoxide dismutase enzymes catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. The process can be viewed as two half reactions, the first generating oxygen, the second generating hydrogen peroxide. The hydrogen peroxide can then itself be detoxified by the action of catalase or peroxidase enzymes.
Role of Salmonella SodC during infection
Salmonella possess two distinct periplasmic SodC enzymes
SodC is induced within macrophages
SodC induction is mediated by PhoPQ
This study examined the two SodC enzymes of Salmonella to assess which plays the dominant role during infection. Reporter genes were used to measure the level of both enzymes. Whilst both enzymes are induced within macrophages and within the spleen during infection of mice, the level of induction of SodCI is considerably greater than that for SodCII.
The second experiment shown here looked at the role of PhoPQ TCS in activating SodCI. PhoPQ system activates genes in response to growth at low magnesium concentrations, so this experiment studied SodCI activity at high (10mM) and low (10uM) Mg concentrations. In a wildtype Salmonella, SodCI was induced by growth in low magnesium, consistent with the role of PhoP-PhoQ. In Salmonella which had a mutated form of PhoP (which was thus inactive), we do not see any such induction of SodCI. In contrast, if we examine SodCI activity in a Salmonella strain that has a constitutively-activated PhoQ protein, we see enormous induction of SodCI irrespective of the magnesium concentration.
Therefore, PhoPQ system mediates the upregulation of SodCI. So not only does PhoPQ confer LPS modifications that help protect against antimicrobial peptides, it also boosts the bacteria’s oxidative stress response.
The pivotal role of the PhoPQ & PmrAB TCSs
During infection, activation of PhoPQ &PmrAB promotes:
Enhanced bile resistance
Enhanced expression of SOD enzymes promoting resistance to oxidative killing
Lipid A modifications that promote resistance to antimicrobial peptides
In addition, they also promote other elements of the “acid tolerance response”
These simple TCSs result in a coordinated bacterial response that is critical for virulence
The role of PhoPQ in Salmonella virulence
phoP/phoQ-deficient Salmonella typhimurium are attenuated for virulence & macrophage survival, and can be used as live vaccines
How does PhoQ respond to multiple stimuli?
Low Mg2+ concentrations result in loss of bridges = ACTIVATION
Acidic pH alters conformation of the periplasmic domain = ACTIVATION
Positively-charged antimicrobial peptides displace Mg2+ = ACTIVATION
Magnesium ions form a bridge between the negatively-charged membrane and a negatively-charged domain of the PhoQ protein. Low magnesium concentrations, acidic pH and the presence of antimicrobial peptides can all impact on this magnesium bridge, leading to PhoQ activation.
Investigating the stimulus for PhoP-PhoQ activation
In vitro, PhoP-PhoQ is inhibited by magnesium at mM range and activated at µM range
Genes activated by PhoP-PhoQ include those for Mg2+ transport
Hypothesis:
Phagolysosomes have low Mg2+ concentrations, thus the PhoP-PhoQ system is activated following phagocytosis
PhoP-PhoQ activation induces Mg2+ transport mechanisms & LPS modifications that promote intracellular survival
Role for pH in PhoP-PhoQ activation in vivo
Used green fluorescent protein (GFP) under the control of PhoPQ to study the timescale of PhoPQ activation
Used green fluorescent protein (GFP) under the control of PhoPQ to study the timescale of PhoPQ activation
A very elegant study suggests that the dominant sugnal for PhoP-PhoQ activation in vivo is acidic pH, rather than low magnesium concentrations. The gene encoding green fluorescent protein was fused to the promoter for phoPQ. When the PhoP-PhoQ system was activated, cells fluoresced greed. They combined this approach with very sensitive measurements of magnesium concentration and pH within macrophages, and observed that GFP induction correlated with a drop in pH and not a decrease in magnesium concentrations.
Conclusions: Two-component systems
Two-component systems typically comprise a membrane-spanning sensor kinase and a cytoplasmic response regulator
They facilitate the adaptive response of bacteria to environmental stimuli and frequently play a key role in virulence
Many TCSs can co-exist within a bacterial cell, but the interaction between sensor kinase and response regulator is very specific
In many cases, the precise stimulus for in vivo activation remains to be determined
