Bacterial virulence and adaptation mediated by two-compnent system signalling

University dissertation from Stockholm : Karolinska Institutet, Microbiology and Tumor Biology Center (MTC)

Abstract: One of the most fundamental ways of signal perception and propagation is mediated by the bacterial two-component system (TCS). These types of systems mediate bacterial adaptation that may cause disease in a host due to expression of virulence factors. Understanding the principles behind the bacterial adaptation process mediated by TCSs may thus aid in the development of novel types of antibiotics. In this work we first characterized the impact of the BarA-UvrY TCS when it comes to adaptation of E. coli to varying carbon sources both in vitro and in a monkey cystitis infection model. Growth and efficient utilization of limited nutrients is central for the bacteria when it comes to the infection process. A bacterium mutated in the BarA-UvrY TCS is deficient in the ability to switch between different carbon sources and thus severely attenuated when nutrients are limited and vary over time. However, when gluconeogenic carbon sources are present in excess our results indicate that lack of the BarA-UvrY TCS leads to a higher fitness, probably due to an increased amount of free CsrA protein promoting gluconeogensis. To get more insight regarding the molecular function of the BarA-UvrY TCS we benefited from the ability of UvrY to be activated by acetyl phosphate. This enabled us to characterize BarA sensor proteins with mutations in domains believed to be important for sensor signal propagation and phosphatase activity. The results indicate that the HAMP domain (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) in BarA is vital for the kinase and phosphatase switching ability. of the sensor and that the two N-terminal domains of BarA are both involved in dephosphorylating UvrY. The dephosphorylating process was also shown to be mediated by a dimer of two BarA sensor proteins. The BarA-UvrY TCSs controls the carbon storage regulator (Csr) system that has earlier been implicated in biofilm formation in both E. coli and Salmonella. We decided to establish a model for studying the kinetics and the impact of these and other regulatory systems on early Salmonella biofilm formation, using a combination of atomic force microscopy and light microscopy. Following the initial proliferation phase, we observed a dispersal of the bacteria and formation of microcolonies that subsequently merged into a confluent biofilm. The dispersal was clearly distinct from the detachment phase that occurs after formation of the mature biofilm. Mutations in different global regulatory genes and genes controlling the production of extracellular polymeric substances (EPS) had a moderate to severe impact on the ability of Salmonella to form a biofilm. Loss of the CsrA protein had a drastic effect on cell morphology and caused a loss of EPS and flagella, unlike loss of the CsrB and CsrC ncRNAs, which caused an increase in EPS and flagella production. In the last study the yhdA gene was identified, via a transposon screening approach, as a factor affecting BarA-UvrY TCS signaling. The yhdA gene encodes a 646 amino acid protein containing both a GGDEF-like and an EAL-like domain. These domains are involved in formation and breakdown of 3',5'-cyclic diguanylic acid (c-di-GMP), a second messenger important for the switch between the vegetative and sessile phase of the bacteria. Additional complementation studies using the yhdA gene expressed in trans, and the ability of UvrY to be activated by acetyl phosphate in the absence of BarA, suggested that YhdA affects the ability of the BarA sensor to switch between phosphatase and kinase activity.

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