The Rpf/DSF system and the regulation of virulence in the plant pathogen Xanthomonas campestris

The full virulence of Xanthomonas campestris pv. campestris (Xcc) to plants depends upon cell-to-cell signalling mediated by the signal molecule DSF (for diffusible signal factor), that has been characterised as cis-11-methyl-2-dodecenoic acid. DSF-mediated signalling regulates motility, biofilm dynamics and the synthesis of particular virulence determinants. The synthesis and perception of the DSF signal molecule involves products of the rpf (regulation of pathogenicity factors) gene cluster. DSF synthesis is fully dependent on RpfF, which encodes a putative enoyl-CoA hydratase. A two-component system, comprising the complex sensor histidine kinase RpfC and the HD-GYP domain regulator RpfG, is implicated in DSF perception. The HD-GYP domain of RpfG is a phosphodiesterase working on cyclic di-GMP; DSF perception is thereby linked to the turnover of this intracellular second messenger. The full range of regulatory influences of the Rpf/DSF system and of cyclic di-GMP in Xcc has yet to be established. In order to further characterise the Rpf/DSF regulatory network in Xcc, a proteomic approach was used to compare protein expression in the wildtype and defined rpf mutants. This work shows that the Rpf/DSF system regulates a range of biological functions that are associated with virulence and biofilm formation but also reveals new functions mediated by DSF regulation. These functions include antibiotic resistance, detoxification and stress tolerance. Mutational analysis showed that several of these regulated protein functions contribute to virulence in Chinese radish. Interestingly, it was demonstrated that different patterns of protein expression are associated with mutations of rpfF, rpfC and rpfG. This suggests that RpfG and RpfC have broader roles in regulation other than perception and transduction of DSF. Taken together, this analysis indicates the broad and complex regulatory role of Rpf/DSF system and identifies a number of new functions under Rpf/DSF control, which were shown to play a role in virulence.

A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Molecular analysis of virulence mechanisms associated with adherent-invasive Escherichia coli (AIEC)

Crohn's Disease (CD) is a chronic inflammatory bowel disease of unknown etiology. Recent work has shown that a new pathotype of Escherichia coli, Adherent Invasive E. coli (AIEC) may be associated with CD. AIEC has been shown to adhere to and invade epithelial cells and to replicate within macrophages (together this is called the AIEC phenotype). In this thesis, the AIEC phenotype of 84 E. coli strains were determined in order to identify the prevalence of this phenotype within the E. coli genus. This study showed that a significant proportion of E. coli strains (approx. 5%) are capable of adhering to and invading epithelial cells and undergoing intramacrophage replication. Moreover, the results presented in this study indicate a correlation between survival in macrophage and resistance to grazing by amoeba supporting the coincidental evolution hypothesis that resistance to amoebae could be a driving force in the evolution of pathogenicity in some bacteria, such as AIEC. In addition, this study has identified an important regulatory role for the CpxA/R two component system (TCS) in the invasive abilities of AIEC HM605, a colonic mucosa-associated CD isolate. A mutation in cpxR was shown to be defective in the invasion of epithelial cells and this defect was shown to be independent of motility or the expression of Type 1 fimbriae, factors that have been shown to be involved in the invasion of another strain of AIEC, isolated from a patient with ileal CD, called LF82. The CpxA/R TCS responds to disturbances in the cell envelope and has been implicated in the virulence of a number of Gram negative pathogens. In this study it is shown that the CpxA/R TCS regulates the expression of a potentially novel invasin called SinH. SinH is found in a number of invasive strains of E. coli and Salmonella. Moreover work presented here shows that a critical mechanism underpinning AIEC persistence in macrophages is the repair of DNA bases damaged by macrophage oxidants. Together these findings provide evidence to suggest that AIEC are a diverse group of E. coli and possess diverse molecular mechanisms and virulence factors that contribute to the AIEC phenotype. In addition, AIEC may have gone through different evolutionary histories acquiring various molecular mechanisms ultimately culminating in the AIEC phenotype. The gastrointestinal (GI) tract harbors a diverse microbiota; most are symbiotic or commensal however some bacteria have the potential to cause disease (pathobiont). The work presented here provides evidence to support the model that AIEC are pathobionts. AIEC strains can be carried as commensals in healthy guts however, when the intestinal homeostasis is disrupted, such as in the compromised gut of CD patients, AIEC may behave as opportunistic pathogens and cause and/or contribute to disease by driving intestinal inflammation.