Subversion of Rho GTPases by WxxxE effectors of attaching and effacing pathogens

Enteropathogenic E. coli (EPEC), Enterohaemorhagic E. coli (EHEC) and Citrobacter rodentium are constituent members of the attaching and effacing (A/E) pathogens. The A/E group of bacteria are considered to be extracellular pathogens which form characteristic lesions by intimately adhering to host enterocytes and directing the effacement intestinal brush border. EPEC and EHEC are diarrhoeal pathogens, which are a global health burden in developing and industrialised countries respectively. Citrobacter rodentium is a murine pathogen which is an excellent animal model for EPEC and EHEC infection. EPEC, EHEC and C. rodentium conserve a genomic region termed the locus of enterocyte effacement (LEE) which encodes a type 3 secretion system (T3SS), a core set of type 3 secreted effector proteins and the outer membrane adhesin intimin, which are essential for A/E lesion formation. A/E pathogens utilise their T3SSs to translocate dozens of effector proteins directly from the bacteria into host cells. Once translocated these effector proteins modulate a range of eukaryotic signalling pathways including those which regulate the host cell cytoskeleton. An example of this is the T3SS effector Tir which localises to the mammalian plasma membrane, acts as a receptor for intimin and subsequently directs the polymerisation of actin rich pedestals beneath adherent bacteria. Subversion of the eukaryotic cytoskeleton is a strategy employed by a range of bacterial pathogens. Due to the pivotal role of Rho GTPases in regulating actin dynamics they are commonly targeted by bacterial virulence factors. Recently a family of type 3 secreted effector proteins has been defined based on their homology around an invariant tryptophan and glutamic acid residue separated by three variable amino acids (WxxxE). In this study we have identified the EspM family of proteins and EspT as novel WxxxE effectors in the A/E pathogens. We demonstrate that the EspM proteins and EspT are translocated into host cells in a T3SS dependent manner. Once translocated, EspM proteins direct the nucleation of actin stress fibres, while EspT drives the formation of lamellipodia and membrane ruffles. Furthermore, we found that the cytoskeletal re-arrangements associated with the WxxxE proteins are dependent on the activation of the small Rho GTPases. EspM proteins activate RhoA while EspT induces the activity of both Rac1 and Cdc42. A more detailed structural and function analysis of the mechanism by which EspM2 activates RhoA revealed that EspM2 binds RhoA in a concentration dependent manner and subsequently promotes RhoA nucleotide exchange from a GDP to GTP bound form. We also show that EspM2 adopts a structure similar to that of the previously reported Salmonella T3SS Rho GEF SopE despite their limited sequence homology. Although the catalytic domain of SopE was not conserved in EspM2 we identified a novel loop which is essential for EspM2 RhoA GEF activity. As expression of EspT resulted in the formation of membrane ruffles which are often associated with the invasion of bacterial pathogens we investigated whether EspT dependent cytoskeletal remodelling could facilitate the internalisation of the canonically non-invasive A/E pathogens. Interestingly, we found that EPEC strains expressing EspT were significantly more invasive than those which did not and furthermore that this invasion was dependent upon the activity of Rac1 and Wave2. Additionally, we demonstrate that once internalised EPEC is maintained within a vacuole (ECV) and is capable of surviving and replicating intracellularly. We also report that EPEC translocates Tir into the vacuolar membrane where it can nucleate actin in an analogous manner to the formation of pedestals by extracellular bacteria. This is the first time an intracellular bacterial pathogen has been shown to polymerise actin tails across a vacuolar membrane. Together the results presented in this study demonstrate that the EspM and EspT families of WxxxE effector proteins are potent modulators of eukaryotic GTPase signalling cascades and as a result convey novel virulence attributes to the A/E pathogen group

The T3SS effector EspT defines a new category of invasive enteropathogenic E. coli (EPEC) which form intracellular actin pedestals.

Enteropathogenic Escherichia coli (EPEC) strains are defined as extracellular pathogens which nucleate actin rich pedestal-like membrane extensions on intestinal enterocytes to which they intimately adhere. EPEC infection is mediated by type III secretion system effectors, which modulate host cell signaling. Recently we have shown that the WxxxE effector EspT activates Rac1 and Cdc42 leading to formation of membrane ruffles and lamellipodia. Here we report that EspT-induced membrane ruffles facilitate EPEC invasion into non-phagocytic cells in a process involving Rac1 and Wave2. Internalized EPEC resides within a vacuole and Tir is localized to the vacuolar membrane, resulting in actin polymerization and formation of intracellular pedestals. To the best of our knowledge this is the first time a pathogen has been shown to induce formation of actin comets across a vacuole membrane. Moreover, our data breaks the dogma of EPEC as an extracellular pathogen and defines a new category of invasive EPEC

Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) translocate dozens of type III secretion system effectors, including the WxxxE effectors Map, EspM and EspT that activate Rho GTPases. While map, which is carried on the LEE pathogenicity island, is absolutely conserved among EPEC and EHEC strains, the prevalence of espM and espT is not known. Here we report the results of a large screen aimed at determining the prevalence of espM and espT among clinical EPEC and EHEC isolates. The results suggest that espM, detected in 51 % of the tested strains, is more commonly found in EPEC and EHEC serogroups that are linked to severe human infections. In contrast, espT was absent from all the EHEC isolates and was found in only 1.8 % of the tested EPEC strains. Further characterization of the virulence gene repertoire of the espT-positive strains led to the identification of a new ζ2 intimin variant. All the espT-positive strains but two contained the tccP gene. espT was first found in Citrobacter rodentium and later in silico in EPEC E110019, which is of particular interest as this strain was responsible for a particularly severe diarrhoeal outbreak in Finland in 1987 that affected 650 individuals in a school complex and an additional 137 associated household members. Comparing the protein sequences of EspT to that of E110019 showed a high level of conservation, with only three strains encoding EspT that differed in 6 amino acids. At present, it is not clear why espT is so rare, and what impact EspM and EspT have on EPEC and EHEC infection

Citrobacter rodentium is an unstable pathogen showing evidence of significant genomic flux.

Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen. In this study, through PFGE, sequencing of isolates showing variation, whole genome transcriptome analysis and examination of the mobile genetic elements, we found that, consistent with our previous hypothesis, the genome of C. rodentium is unstable as a result of repeat-mediated, large-scale genome recombination and because of active transposition of mobile genetic elements such as the prophages. We sequenced an additional C. rodentium strain, EX-33, to reveal that the reference strain ICC168 is representative of the species and that most of the inactivating mutations were common to both isolates and likely to have occurred early on in the evolution of this pathogen. We draw parallels with the evolution of other bacterial pathogens and conclude that C. rodentium is a recently evolved pathogen that may have emerged alongside the development of inbred mice as a model for human disease

Quantifying the response of the ORAC aerosol optical depth retrieval for MSG SEVIRI to aerosol model assumptions

We test the response of the Oxford-RAL Aerosol and Cloud (ORAC) retrieval algorithm for MSG SEVIRI to changes in the aerosol properties used in the dust aerosol model, using data from the Dust Outflow and Deposition to the Ocean (DODO) flight campaign in August 2006. We find that using the observed DODO free tropospheric aerosol size distribution and refractive index increases simulated top of the atmosphere radiance at 0.55 µm assuming a fixed erosol optical depth of 0.5 by 10–15 %, reaching a maximum difference at low solar zenith angles. We test the sensitivity of the retrieval to the vertical distribution f the aerosol and find that this is unimportant in determining simulated radiance at 0.55 µm. We also test the ability of the ORAC retrieval when used to produce the GlobAerosol dataset to correctly identify continental aerosol outflow from the African continent and we find that it poorly constrains aerosol speciation. We develop spatially and temporally resolved prior distributions of aerosols to inform the retrieval which incorporates five aerosol models: desert dust, maritime, biomass burning, urban and continental. We use a Saharan Dust Index and the GEOS-Chem chemistry transport model to describe dust and biomass burning aerosol outflow, and compare AOD using our speciation against the GlobAerosol retrieval during January and July 2006. We find AOD discrepancies of 0.2–1 over regions of intense biomass burning outflow, where AOD from our aerosol speciation and GlobAerosol speciation can differ by as much as 50 - 70 %

Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters Rho1 Activity on Peroxisomes

SifA is a virulence protein required for assembly and tubulation of a modified phagosome that promotes Salmonella replication. We show that SifA expressed in yeast induces membrane invagination during peroxisome proliferation and requires functional Rho1p. This is consistent with SifA ability to interact with RhoA and the fact that it is a GEF structural homologue

Improved understanding of aerosol processes using satellite observations of aerosol optical properties

Atmospheric aerosols are the largest remaining uncertainty in the Earth’s radiative budget and it is important that we improve our knowledge of aerosol processes if we are to understand current radiative forcing and accurately project changes in future climate. Aerosols affect the radiation balance directly through the absorption and scattering of incoming solar radiation and indirectly through the modification of cloud microphysical properties. Understanding aerosol forcing remains challenging due to the short atmospheric residence time of aerosols resulting in large spatial and temporal heterogeneity in aerosol loading and chemical composition. Satellite retrievals are becoming increasingly important to improving our knowledge of aerosol forcing. They provide regular global data at finer spatial and temporal resolution than available through sparse groundbased point measurements or localised aircraft campaigns, but cannot unambiguously determine aerosol speciation, relying heavily on a priori assumptions. In this thesis I use data from two satellite instruments: the Along Track Scanning Radiometer 2 (ATSR-2) and the Spinning Enhanced Visible and InfraRed Imager (SEVIRI) interpreted using the Oxford-RAL Aerosol and Cloud (ORAC) retrieval scheme in three pieces of interrelated work. First I use satellite observations of aerosol optical depth a and cloud particle effective radius re from the ATSR-2 instrument in 1997 to investigate the Twomey indirect effect (IE, -δ ln re /δ ln τa) in regions of continental outflow. I generally find a negative correlation between τa and re with the strongest inverse relationships downwind of Africa. North America and eastern Asian continental outflow exhibits a strong seasonal dependence, as expected. Global values for IE range from 0.10 to 0.16, consistent with theoretical predictions. Downwind of Africa, I find that the IE is unphysically high but robust (r = −0.85) during JJA associated with high aerosol loading, and attribute this tentatively to the Twomey hypothesis accounting only for a limited number of physical properties of aerosols. Second, I test the response of the Oxford-RAL Aerosol and Cloud (ORAC) retrieval algorithm for MSG SEVIRI to changes in the aerosol properties used in the dust aerosol model, using data from the Dust Outflow and Deposition to the Ocean (DODO) flight campaign in August 2006. I find that using the observed DODO free tropospheric aerosol size distribution and refractive index compared with the dust aerosol properties from the Optical Properties of Aerosol and Cloud (OPAC) package, increases simulated top of the atmosphere radiance at 0.55 μm assuming a fixed aerosol optical depth of 0.5, by 10–15%, reaching a maximum difference at low solar zenith angles. This difference is sensitive to changes in AOD, increasing by ~2–4% between AOD of 0.4–0.6. I test the sensitivity of the retrieval to the vertical distribution of the aerosol and find that this is unimportant in determining simulated radiance at 0.55 μm. I also test the ability of the ORAC retrieval when used to produce the GlobAerosol dataset to correctly identify continental aerosol outflow from the African continent and I find that it poorly constrains aerosol speciation. I develop spatially and temporally resolved prior distributions of aerosols to inform the retrieval which incorporates five aerosol models: desert dust, maritime, biomass burning, urban and continental. I use a Saharan Dust Index and the GEOS-Chem chemistry transport model to describe dust and biomass burning aerosol outflow, and compare AOD using my speciation against the GlobAerosol retrieval during January and July 2006. I find AOD discrepancies of 0.2–1 over regions of biomass burning outflow, where AOD from my aerosol speciation and the GlobAerosol speciation can differ by as much as 50 - 70 %. Finally I use satellite observations of aerosol optical depth and cloud fraction from the MSG SEVIRI instrument to investigate the semi-direct effect of Saharan dust aerosol on marine stratocumulus cloud cover over the Atlantic during July 2006. I first use these data to study the spatial autocorrelation of aerosol optical depth and find that it is correlated over a lag of 0.1◦ (approximately 10 km at low latitudes), beyond which it rapidly decorrelates. I find a 15 % higher cloud fraction in regions with high dust loading (AOD > 0.5), compared with scenes with a lower dust loading (AOD < 0.5), which for high dust scenes increases with local static stability. I attribute this tentatively to aerosol solar shielding enhancing longwave cloud top radiative cooling which drives marine stratocumulus convection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors▿

Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection