Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Access control : allocating resources to selfish agents

The ultimate goal of an authorisation system is to allocate each user the level of access they need to complete their job - no more and no less. This proves to be challenging in an organisational setting because on one hand employees need enough access to perform their tasks, while on the other hand more access will bring about an increasing risk of misuse - either intentionally, where an employee uses the access for personal benefit, or unintentionally through carelessness, losing the information or being socially engineered to give access to an adversary. With the goal of developing a more dynamic authorisation model, we have adopted a game theoretic framework to reason about the factors that may affect users’ likelihood to misuse a permission at the time of an access decision. Game theory provides a useful but previously ignored perspective in authorisation theory: the notion of the user as a self-interested player who selects among a range of possible actions depending on their pay-offs.

Ontology-based specific and exhaustive user profiles for constraint information fusion for multi-agents

Intelligent agents are an advanced technology utilized in Web Intelligence. When searching information from a distributed Web environment, information is retrieved by multi-agents on the client site and fused on the broker site. The current information fusion techniques rely on cooperation of agents to provide statistics. Such techniques are computationally expensive and unrealistic in the real world. In this paper, we introduce a model that uses a world ontology constructed from the Dewey Decimal Classification to acquire user profiles. By search using specific and exhaustive user profiles, information fusion techniques no longer rely on the statistics provided by agents. The model has been successfully evaluated using the large INEX data set simulating the distributed Web environment.

Property Agents and Motor Dealers Act : strike three

The Tourism, Racing and Fair Trading (Miscellaneous Provisions) Act 2002 (“the Act”) which was passed on 18 April 2002 contains a number of significant amendments relevant to the operation of the Property Agents and Motor Dealers Act 2000. The main changes relevant to property transactions are: (i) Changes to the process for appointment of a real estate agent and consolidation of the appointment forms; (ii) Additions to the disclosure obligation of agents and property developers; (iii) Simplification of the process for commencing the cooling off period; (iv) Alteration of the common law position concerning when the parties are bound by a contract; (v) Removal of the requirement for a seller’s signature on the warning statement to be witnessed; (vi) Retrospective amendment of s 170 of the Body Corporate and Community Management Act 1997; (vii) Inclusion of a new power to allow inspectors to enter the place of business of a licensee or a marketeer without consent and without a warrant; and (viii) Inclusion of a new power for inspectors to require documents to be produced by marketeers. The majority of the amendments are effective from the date of assent, 24 April 2002, however, some of the amendments do not commence until a date fixed by proclamation. No proclamation has been made at the time of writing (2 May 2002). Where the amendments have not commenced this will be noted in the article. Before providing clients with advice, practitioners should carefully check proclamation details.

Property Agents and Motor Dealers Act : take two

The Property Agents and Motor Dealers Act 2000 commenced on 1 July 2001. Significant changes have now been made to the Act by the Property Agents and Motor Dealers Amendment Act 2001 (“the amending Act”). The amending Act contains two distinct parts. First, ss 11-19 of the amending Act provide for increased disclosure obligations on real estate agents, property developers and lawyers together with an extension of the 5 business day cooling-off period imposed by the original Act to all residential property (other than contracts formed on a sale by auction). These provisions commenced on 29 October 2001. The remaining provisions of the amending Act provide for increased jurisdiction and powers to the Property Agents and Motor Dealers Tribunal (“the Tribunal”) enabling the Tribunal to deal with claims against marketeers. These provisions commenced on the date of assent, 21 September 2001.

Real estate agents, brochure and misleading or deceptive conduct

The decision of the High Court in Butcher v Lachlan Elder Realty Pty Ltd [2004] HCA 60 involves issues that affect every person who is induced to buy real estate in Australia by statements in sales brochures distributed by real estate agents. One of these issues is the extent to which estate agents unwittingly engage in misleading or deceptive conduct under s 52 of the Trade Practices Act 1974 (Cth) (‘the Act’) when they distribute sales brochures that contain untrue or misleading statements prepared by others. A further issue is the extent to which agents can escape liability by relying on disclaimers about the authenticity of false statements contained in brochures prepared by them.

Waiver of statutory requirements : Property Agents and Motor Dealers Act 2000 (Qld)

One of the many difficulties associated with the drafting of the Property Agents and Motor Dealers Act 2000 (Qld) (‘the Act’) is the operation of s 365. If the requirements imposed by this section concerning the return of the executed contract are not complied with, the buyer and the seller will not be bound by the relevant contract and the cooling-off period will not commence. In these circumstances, it is clear that a buyer’s offer may be withdrawn. However, the drafting of the Act creates a difficulty in that the ability of the seller to withdraw from the transaction prior to the parties being bound by the contract is not expressly provided by s 365. On one view, if the buyer is able to withdraw an offer at any time before receiving the prescribed contract documentation the seller also should not be bound by the contract until this time, notwithstanding that the seller may have been bound at common law. However, an alternative analysis is that the legislative omission to provide the seller with a right of withdrawal may be deliberate given the statutory focus on buyer protection. If this analysis were correct the seller would be denied the right to withdraw from the transaction after the contract was formed at common law (that is, after the seller had signed and the fact of signing had been communicated to the buyer).

Property Agents and Motor Dealers Act (Qld) : disclosure, cooling-off and marketeers

The Property Agents and Motor Dealers Act 2000 commenced on 1 July 2001. Significant changes have now been made to the Act by the Property Agents and Motor Dealers Amendment Act 2001 (“the amending Act”). The amending Act contains two distinct parts. First, ss 11-19 of the amending Act provide for increased disclosure obligations on real estate agents, property developers and lawyers together with an extension of the 5 business day cooling-off period imposed by the original Act to all residential property (other than contracts formed on a sale by auction). These provisions are expected to commence on 29 October 2001. The remaining provisions of the amending Act provide for increased jurisdiction and powers to the Property Agents and Motor Dealers Tribunal (“the Tribunal”) enabling the Tribunal to deal with claims against marketeers. These provisions commenced on the date of assent (21 September 2001).

Computing with motile bio-agents

We describe a model of computation of the parallel type, which we call 'computing with bio-agents', based on the concept that motions of biological objects such as bacteria or protein molecular motors in confined spaces can be regarded as computations. We begin with the observation that the geometric nature of the physical structures in which model biological objects move modulates the motions of the latter. Consequently, by changing the geometry, one can control the characteristic trajectories of the objects; on the basis of this, we argue that such systems are computing devices. We investigate the computing power of mobile bio-agent systems and show that they are computationally universal in the sense that they are capable of computing any Boolean function in parallel. We argue also that using appropriate conditions, bio-agent systems can solve NP-complete problems in probabilistic polynomial time.