What are two component regulatory systems (TCRS)? Bacteria respond to changing environments by altering gene expression. Two-component regulatory systems (TCRS) are important mediators of signal transduction that enable bacteria to detect physical and/or chemical changes and then relay this signal through the cytoplasm to the bacterial nucleoid, where modulation of gene expression occurs. In its most basic form, a TCRS consists of a membrane-bound sensor kinase and a DNA-binding response regulator. In response to specific stimuli, the sensor kinase is phosphorylated at a conserved histidine residue and then the phosphoryl group is transferred to the conserved aspartate on the response regulator. Phosphorylation of the response regulator triggers a conformational change, driving dimerization and high affinity DNA binding. For example, the SsrA sensor kinase in Salmonella is activated by low pH and phosphorylates the response regulator SsrB. Phosphorylated SsrB de-represses H-NS silencing and activates a set of virulence genes located at the Salmonella Pathogenicity island-2 (SPI-2) region. These genes encode various components of a molecular syringe (Type-III secretion system) which allows the delivery of ~30 specialized effector proteins that manipulate the host cellular machinery to ensure the survival and proliferation of intracellular Salmonella.
After bacteria enter the host cell, they often form vacuoles that enable them to survive, reproduce, and spread infection. This occurs in both phagocytic and non-phagocytic host cells. The Salmonella Containing Vacuole (SCV) is integrated with the early endocytic pathway, but these vacuoles are able to escape lysosomal fusion and lysis. During SCV maturation, an F-actin meshwork is formed around bacterial vacuoles in a process known as vacuole-associated actin polymerization (VAP) that reinforces the integrity of the vacuolar membrane. Mature SCVs are found in a perinuclear position, proximal to the Golgi apparatus. Salmonellae within SCVs also induce the formation of tubular aggregates along a scaffold of microtubules called Salmonella-induced filaments (SIFs) that extend from SCVs throughout the cell. Therefore, an intricate link exists between the host cytoskeleton and Salmonella pathogenesis at various stages. Other intracellular bacteria like Mycobacteria, Coxiella, Legionella, and Brucella also reside within vacuoles and exploit different components of the endocytic and secretory pathways for pathogenesis.
Bacterial cytoplasmic pH can be measured in single cells using the I-switch biosensor labeled at its 5′ and 3′ termini with Alexa-488 and Alexa-647, two different fluorescent molecules. Bacteria are electroporated with the I-Switch The I–switch consists of cytosine-rich unpaired regions that form an anti-tetraplex CH+.C by alternate Watson and Crick base pairing in the presence of protons. This leads to a “closed” conformation of DNA, enabling FRET to occur between the fluorophore pairs. This process is reversible, and at neutral pH, the I-switch dissociates into an open, extended conformation because of electrostatic repulsion between the duplex arms. The FRET intensities provide a measure of the cytoplasmic pH in bacteria.