Genetic reductionist approach for studing the two-component signaling system in Staphylococcus aureus
Authors:   , Villanueva San Martín Maite , Toledo Arana Alejandro 
Published: March 2022

Staphylococcus aureus is a Gram-positive bacterium adapted to live in a wide variety of environmental niches. In humans, S. aureus colonizes the nose, skin, axilla, groin, throat or intestinal tract. Approximately 20% of the anterior nares of healthy human adults are persistently colonized with S. aureus. In some circumstances, the bacterium is able to traverse the epithelial barrier reaching internal organs. When this occurs, S. aureus can cause a variety of diseases, ranging from minor skin infections, such as furuncles or abscesses, to severe infections, such as endocarditis, osteomyelitis, pneumonia or toxic shock syndrome. S. aureus needs to recognize environmental signals, transmit stimuli to the cytoplasm and activate the necessary changes to adapt the bacterial physiology to the conditions of each environmental niche. The main mechanism to sense and respond to environmental signals in bacteria is the two-component transduction system (TCSs). TCSs comprise a membrane sensor histidine kinase (HK) and a cytoplasmic response regulator (RR). During the activation process, the sensor receives a specific signal and autophosphorylates itself on a conserved histidine residue. The phosphate is then transferred to an aspartate residue of the cytoplasmic cognate RR. The phosphorylated RR triggers a specific response in accordance with the signal. The genome of a single bacterial species usually encodes for multiple signal transducers: the number often proportional to the genome size, the diversity of environments in which organisms live and the complexity in cellular differentiation. Bacteria inhabiting relatively stable host environments, such as obligate intracellular parasites, encode for few or even no TCS signalling systems, while ubiquitous bacteria that are able to live in a variety of environments encode high numbers of TCSs. In relation with the number and the function of TCSs in bacteria, several questions remain open: How many TCSs does a free-living bacterium need to live? Are TCSs necessary when bacteria live in a constant environment? Does cross-activation between different TCSs exist in vivo? With the aim to answer these questions and to carry out a global analysis of the cellular processes controlled by TCSs, we generated S. aureus mutants devoid of the TCS signalling networks by using a genetic reductionist approach on two genetically unrelated S. aureus strains. The work consisted in the sequential deletion of the 15 non-essential TCSs of S. aureus as well as the deletion of the sensor (WalK) of the walKR TCS, whose complete deletion is lethal. The resulting mutants lacking the TCSs demonstrate that under constant environmental conditions these systems are dispensable for S. aureus survival. Phenotypic analyses of the mutants devoid of TCSs revealed growth levels indistinguishable from the wild type at 37 and 44ºC, and similar metabolic capacities. However, mutants devoid of TCSs lose the capacity to reduce nitrate to nitrite, show lower growth rates at 28ºC and capacity to survive in the environment and higher sensitivity to detergents. Moreover, in the absence of TCSs, S. aureus is unable to invade eukaryotic cells and colonize organs, rendering the bacteria avirulent in a mouse infection model. Phenotypes associated to the TCS-deficient mutant can be restored by the ectopic expression of single TCSs, indicating that each phenotype is most likely modulated by a single TCS. The TCS-deficient strain was then used as a platform for studying signal transduction specificity (cross-talk) in vivo. For that purpose, we developed a simple method based on the complementation of the TCSdeficient mutant with plasmids that containing a combination of the HK family and a RR. Analysis of these complemented strains allowed the identification of cross-talk between GraS and ArlR. The cross-talk occurs even in the presence of the corresponding ArlS HK and GraR RR. Taking into account that our systematic analysis only found cross-activation between these two TCSs, we conclude that cross-activation between TCSs can occur in vivo, but it is rare. We anticipate that the strains lacking the TCSs, or the set of strains containing single TCSs, will be extremely useful to identify the regulon of each TCS or for finding antimicrobials that specifically block TCS functions.