L3 - Microbial sensing of the host environment (TCSs) Flashcards
The importance of niche & host adaptation: The environmental cues
ENVIRONMENTAL CUES INCLUDE: Temperature Acidic pH Ions – e.g. Mg2+, Fe3+ Population density (QS) Osmolarity Presence of antimicrobials Oxygen availability Nutrient availability Mammalian hormones
Define two-component systems (TCSs)
Two-component systems (TCSs) promote the adaptive response to environmental stimuli
Although commonly viewed as ‘simple’ systems comprising of single HK and RR proteins, complexity can arise via:
HK-HK interactions
putative cross-talk between non-cognate proteins
linkage to other regulatory networks
Through a phospho-relay system, TCSs facilitate the adaptive response of bacteria to diverse environmental stimuli
The HisKA (dimerization/phosphoacceptor) domain contains a conserved histidine residue and is activated via trans-autophosphorylation by the catalytic domain of the histidine kinase The HATPase domain harbours the ATP binding site
The histidine kinase (HK) is transmembrane, with the sensing domain located outside of the plasma membrane, flanked by two transmembrane domains. The response regulator is a cytoplasmic protein.
Sensing of stimulus by the HK leads to trans-autophosphorylation of the conserved histidine
Subsequent phosphotransfer occurs to the conserved aspartate on the receiver (Rec) domain of the RR
Phosphorylated forms of RR proteins typically have a short half-life
Many RRs have autophosphatase activity
Many HKs also possess phosphatase activity towards their RR
Typically, the response regulator is a DNA-binding protein that influences (positively or negatively) the expression of target genes. These changes in gene expression facilitate the adaptive response to the original stimulus that was sensed by the HK.
Phosphorylation of the RR alters the activity of the protein
The output domain is typically a DNA-binding domain, influencing expression of target genes
The output domain of some RRs have enzymatic activity.
Irrespective of the precise composition of the two-component system, the movement of the phosphoryl group is always H → D →H → D etc.
Specificity of HK-RR interactions in TCS is essential. How is specificity achieved?
Multiple TCSs exist within individual bacterial cells – some species have in excess of 100 different HKs.
“Specificity residues” (coevolving amino acids in the HK & RR proteins) determine which REC domains the kinases can phosphorylate
Typically, kinases from a single species share ≤ 3 specificity residues to prevent cross-talk
It is possible to rewire networks by altering specificity residues. Increasingly, the view of TCSs functioning in isolation is being challenged
Define the Multi-kinase networks in Pseudomonas
The GacS network including the closely affiliated HptB and SagS/BfiS branches. Red ovals show SKs, blue ovals show RRs, the purple oval shows the HptB protein and the grey ovals show other proteins in the system. Arrows show stimulatory interactions, while blunt-ended lines show inhibitory interactions and bulb-ended lines show interactions that can be stimulatory or inhibitory depending on conditions. The primary output of the GacS side of the pathway is the small RNAs RsmY and RsmZ, which sequester the post-transcriptional regulators, RsmA and RsmN. When RsmA and RsmN are sequestered, virulence genes associated with chronic infection are upregulated while those associated with acute virulence genes are downregulated. Conversely, when RsmA and RsmN are free, the acute virulence genes are upregulated and the chronic infection genes are downregulated. The HptB and SagS/BfiS branches of the pathway also regulate RsmY and RsmZ levels, respectively. The role of HsbA differs depending on whether it is phosphorylated (blue arrow) or dephosphorylated (red arrow). Two diguanylate cyclases are controlled by this network, HsbD and SadC (Picture in notes).
What are the conserved HisKA specificity residues in the Gac network (Multi-kinase networks in Pseudomonas)
(Histidine kinase) LadS specificity residues: S G G S E L
(Histidine kinase) GacS specificity residues: N G G F N L
(Histidine kinase) RetS specificity residues: N G G M E L
(Histidine kinase) PA1611 specificity residues: N G G M Q L
What are the four predicted metal-responsive TCSs in Burkholderia cenocepacia?
Zn2+ Co2+ Cd2+ / Cu2+
Heavy metal-responsive systems are implicated in virulence
What is Burkholderia cenocepacia?
an opportunistic pathogen of immunocompromised patients, particularly those with cystic fibrosis
What are the Burkholderia cenocepacia HisKA specificity residues in the metal-responsive TCSs?
NOT highly conserved
(Histidine kinase) IrIs specificity residues: A N S S Q I
(Histidine kinase) SO585 specificity residues: N N G A E V
(Histidine kinase) CzcS specificity residues: T N A A Q V
(Histidine kinase) M1417 specificity residues: S I G Q Q V
What are the Burkholderia cenocepacia Rec domains of the RRs specificity residues in the metal-responsive TCSs?
In contrast, specificity residues in the Rec domains of the RRs are highly conserved:
(Rec domains of RRs) IrIR specificity residues: E K M Y K G A
(Rec domains of RRs) SO586 specificity residues: E K T Y K G A
(Rec domains of RRs) CzcR specificity residues: E K T Y K G D
(Rec domains of RRs) M1418 specificity residues: E K V Y S G S
cross-regulation between metal-responsive TCSs.
Phosphotransfer is evident between the cognate HK-RR proteins
Phosphotransfer assays like that depicted above are used to study the transfer of phosphate from histidine kinase (HK) to response regulator (RR).
In the left-hand side of the figure, the cytoplasmic domain of IrlS is incubated with 32P-ATP. At the indicated timepoints, aliquots are removed, “quenched” (to stop the reaction) and then analysed by SDS-PAGE. As indicated, the IrlS is rapidly phosphorylated, as shown by the accumulation of signal by 1 minute.
In the right-hand side of the figure (“IrlS + IrlR”), the same assay is repeated, but this time with the inclusion of the partner response regulator (IrlR). The RR rapidly takes the phosphate from the HK, with the result that no phosphorylated HK is observed but phosphorylated RR accumulates over time.
In vitro phosphotransfer assays suggest non-cognate transfer is also possible:
Same type of phosphotransfer assays as on the previous slide, but this time examining non-cognate interactions.
The left hand image shows that IrlS can phosphorylate the non-cognate response regulator S0586. Note that it appears to occur more slowly than the cognate transfer on the previous slide, and that the HK (IrlS) retains its phosphorylation status. Together, this implies that non-cognate transfer can occur, but is less favourable.
The question is, can non-cognate transfer still occur in the presence of the normal cognate partner. The right hand image shows that it can. Here, IrlS is incubated with IrlR AND S0586. We can see phosphotransfer to both IrlR (the cognate RR) and S0586 (the non-cognate RR). Note that in this experiment, the IrlR protein had to be tagged with eGFP so that we could tell it apart on the SDS-PAGE gel from S0586 (without the tag, the proteins would be the same size).
What is Salmonella?
Important causes of enteric disease in all vertebrates
Salmonella enterica divided into serotypes, for example:
Salmonella enterica serotype Typhimurium (Salmonella typhimurium)
Salmonella enterica serotype Typhi (Salmonella typhi)
What is the route 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.
What are the innate defences against Salmonella infection in the stomach & gastrointestinal tract?
Acidic pH
Gastric pH can be as low as 1.5
An efficient “acid tolerance response” is critical to promote the survival & growth of microbes
Bile salts
Component of bile; has detergent-like antimicrobial activity
Bacterial resistance is typically based around permeability.
Bile is discharged into the small intestine to aid digestion
To facilitate resistance to bile salts, bacteria decrease porin expression and boost efflux systems in order to minimise their permeability to bile salts. Porins are essentially channels that span the outer membrane of Gram-negative bacteria, forming a pore that allows the entrance and exit of solutes.
What are the innate defences against Salmonella infection in the macrophage?
Acidification of the phagosome
V-ATPase complex translocates H+ across the membrane
Reactive oxygen & nitrogen species
NADPH oxidase & iNOS create ROS and RNS respectively
Antimicrobial peptides
Cationic peptides that bind to the negatively-charged bacterial cell
Sequestration of essential nutrients
e.g. action of metal transporters and metal binding proteins.
The phagosome is the vesicle within phagocytic cells in which the ingested material (e.g. bacterial cell) is found following phagocytosis. The phagosome contains numerous bactericidal activities that aim to kill the ingested bacterium.
The V-ATPase complex used energy derived from hydrolysis of ATP to translocate protons (H+) across the membrane into the phagosome, resulting in acidification. The acidic pH is damaging to the microbe, as well as boosting the activity of enzymes within the phagosome whose function is optimal at acidic pH.
A major killing mechanism is through reactive oxygen species (ROS) that are generated directly or indirectly by the NADPH oxidase complex. The oxidase releases O2(-) into the lumen, which can dismutate to hydrogen peroxide. This can then react with O2(-) to generate hydroxyl radicals, and can also be converted into hypochlorous acid by myeloperoxidase (MPO). Collectively, these ROS are highly toxic, and effectively kill microorganisms.
Reactive nitrogen species are also produced, largely by inducible nitric oxide synthase (iNOS). Nitric oxide is produced by iNOS on the cytoplasmic side of the phagosome, and then diffuses across the membrane into the phagosome. The nitric oxide can then undergo spontaneous or catalytic conversion to a range of RNS including nitrogen dioxide and peroxynitrite. ROS and RNS synergize to exert highly toxic effects.
Antimicrobial peptides are small (12-50 amino acids) peptides with potent antimicrobial activity. They are positively-charged (cationic) and interact with the negatively-charged bacterial membrane. They disrupt the membrane, causing cell death.
All organisms require metal for growth and survival, as metals are essential cofactors for numerous enzymes. The phagosome attempts to starve the ingested bacterium of such metals by either pumping the metals out of the phagosome or by sequestering the metals within the phagosome. The diagram above shows this process for iron, but certain other metals are similarly treated, including manganese.
The PhoP-PhoQ and PmrA-PmrB TCSs of Salmonella
PhoP-PhoQ and PmrA-PmrB TCSs are very well characterized. Within the PhoPQ system, PhoQ is the histidine kinase, and PhoP the response regulator. Within the PmrAB system, PmrB is the histidine kinase and PmrA the response regulator.
Within Salmonella, the PhoPQ and PmrAB systems have a distinctive feature that they are connected by the actions of PmrD – a protein which is activated by the PhoPQ system and then promotes expression of PmrA-regulated genes by protecting the phosphorylated (active) form of PmrA. Ordinarily there is a low basal level of phospho-PmrA, kept low by phosphatase activity of PmrB. PmrD specifically binds to phospho-PmrA, protecting it from phosphatase activity.
This connection via PmrD means that the same set of genes can be activated by a larger set of stimuli.
