Lactobacillus reuteri inhibition of Enteropathogenic Escherichia coli adherence to human intestinal epithelium

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains

The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

Balancing mining and the environment : South Africa’s legal framework concerning pollution caused by mining, with examples from the West Rand and Emalahleni.

Thesis (LLM)--Stellenbosch University, 2023.ENGLISH ABSTRACT: Environmental law is a continually growing sphere of law, both locally and internationally. The increase in the importance and presence of environmental law in South Africa, and on a global scale, is as a result of the impacts that human activities have on the environment. These environmental impacts caused by human activities have resulted in there being an escalation in the need to conserve and protect the environment. Section 24 of the Bill of Rights of the Constitution of South Africa incorporates the principle of environmental protection and sustainable development into the constitutional law of South Africa, and generally recognises the fundamental importance of the environment, whilst the National Environmental Management Act 107 of 1998 and the environmental legislation and measures that have followed its adoption address this right as well as various elements of environmental protection, conservation and mitigation. This research focuses on the environmental elements of water and air, which are both vastly impacted on and polluted by the mining industry.AFRIKAANSE OPSOMMING: Omgewingswet is ‘n area van die gereg wat voordurend uitbrei, beide binnelands en internasionaal. Die groei in die belang en teenwoordigheid van omgewingswet in Suid-Afrika, sowel as in die wêreld, is as gevolg van die impak wat menslike aktiwiteite op die omgewing het. Hierdie impak op die omgewing deur menslike bedrywe het veroorsaak dat daar ‘n massiewe groei in die nood is om die omgewing te beskerm en bewaar. Seksie 24 van die Handves van Regte van die Grondwet van Suid-Afrika sluit die beginsel van omgewings beskerming in, sowel as volhoudbare ontwikkeling in die grondwetlike wet van Suid-Afrika. Die grondwet herken oor die algemeen die fundamentele belang van die omgewing terwyl die Nasionale Omgewings Bestuurswet 107 van 1998, sowel as die omgewings wette en maatreëls wat aangeneem is as gevolg daarvan. Hierdie wette, in kombinasie met die verskeie elemente van omgewings beskerming, bewaring en versagting adresseer omgewings bestuur erken hierdie regte. Hierdie navorsing is gemik op die omgewings bestuur van water en lug, wat albei geaffekteer en besoedel word deur mynbou deur die myn bedryf.Master

Applications of Microscopy in <i>Salmonella </i>Research

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen–host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research

The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease

Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway

Immigration Federalism: A Reappraisal

This Article identifies how the current spate of state and local regulation is changing the way elected officials, scholars, courts, and the public think about the constitutional dimensions of immigration law and governmental responsibility for immigration enforcement. Reinvigorating the theoretical possibilities left open by the Supreme Court in its 1875 Chy Lung v. Freeman decision, state and local offi- cials characterize their laws as unavoidable responses to the policy problems they face when they are squeezed between the challenges of unauthorized migration and the federal government’s failure to fix a broken system. In the October 2012 term, in Arizona v. United States, the Court addressed, but did not settle, the difficult empirical, theoretical, and constitutional questions necessitated by these enactments and their attendant justifications. Our empirical investigation, however, discovered that most state and local immigration laws are not organic policy responses to pressing demographic challenges. Instead, such laws are the product of a more nuanced and politicized process in which demographic concerns are neither neces- sary nor sufficient factors and in which federal inactivity and subfederal activity are related phenomena, fomented by the same actors. This Article focuses on the con- stitutional and theoretical implications of these processes: It presents an evidence- based theory of state and local policy proliferation; it cautions legal scholars to rethink functionalist accounts for the rise of such laws; and it advises courts to reassess their use of traditional federalism frameworks to evaluate these sub federal enactments

Ability of enteroaggregative Escherichia coli strains to adhere in vitro to human intestinal mucosa

A collection of 44 enteroaggregative Escherichia coli (EAggEC) strains isolated from infants with diarrhea in India and the United Kingdom were examined for their ability to adhere in vitro to human intestinal mucosa and by electron microscopy for production of putative adherence factors. None of the strains adhered to human duodenal mucosa, and six strains tested did not adhere to ileal mucosa; all 44 strains, however, adhered to human colonic mucosa in localized aggregates. Electron microscopy of infected colonic mucosa indicated fimbrially mediated adhesion of the EAggEC strains. Four morphologically distinct kinds of fimbriae, including a new morphological type of E. coli fimbriae consisting of bundles of fine filaments, were identified among the EAggEC strains; this new type of fimbria was observed in 43 of the 44 EAggEC strains. Forty-three of the 44 EAggEC strains were positive with a DNA probe developed to identify EAggEC, and most of the strains belonged to serotypes unrelated to the other major classes of diarrheic E. coli. These results suggest that EAggEC may be a large-bowel pathogen and colonize the colon by a fimbrially mediated adhesion mechanism