Escherichia coli (E. coli) Kl is one of the commonest Gram negative bacteria causing neonatal bacterial meningitis in both developed and developing countries. Haematogenous spread is a key step in E. coli Kl meningitis; however, it is not clear how bacteria cross the brain endothelium to gain entry into the central nervous system. Previous studies have focussed mainly on the identification of bacterial virulence factors, as well as the signalling pathways that are activated for the recruitment of actin cytoskeleton to the bacterial adhesion site on the apical surface of human brain microvascular endothelial cells (HBMEC) and finally lead to bacterial uptake. However, the cellular requirements and mechanisms of post-invasion events are poorly understood. This study aims to further characterize E. coli KI entry, intracellular trafficking and the associated molecular mechanisms. To achieve this, a virulent fluorescent proteinexpressing E. coli K I strain was constructed. In a previous study, caveolin-l, a lipid raft marker associated with clathrin-independent endocytosis, was found associated with invading and intracellular bacteria in HBMEC. To further study the effect of caveolin-l on the bacterial entry, different caveolin-l mutants were applied here. Overexpression of caveolin-l Y 14A mutant and caveolin-l~, which is non-phosphorylatable, did not block E. coli Kl invasion of HBMEC. Furthermore, E. coli Kl invasion of caveolin-l knockout mouse lung endothelial cells (MLEC) was not blocked, which suggested that caveolin-l was not required for E. coli K 1 invasion of endothelial cells. The role of dynamin, a large GTPase that has been implicated in the membrane fission of caveolae buds, was also investigated. Based on quantitative microscopy scoring, no evidence of any inhibitory effect on the bacterial invasion was observed in cells overexpressing green fluorescent protein- (GFP) tagged dominant negative dynamin 2 [Dyn2(aa)K44A] and dominant negative dynamin 1 (DynlK44A). The experimental evidence from this study therefore suggests that E. coli Kl might invade HBMEC via a caveolae- and dynamin-independent endocytic pathway. To further explore the endocytosis pathway that the bacteria use to invade HBMEC, immunofluorescence staining of E. coli Kl infected HBMEC revealed colocalization of the bacteria with flotillin 1, another lipid raft marker associated with clathrin-independent endocytosis. However, E. coli K1 infection of flotillin 1 knockout MLEC demonstrated a significantly increased bacterial uptake. This observation suggests that E. coli K 1 uptake does not require flotillin 1. In parallel, the number of intracellular non-pathogenic E. coli K-12 recovered from the lysates of flotillin 1 knockout MLEC was also significantly higher than that recovered from the lysates of wild type MLEC. Further, overexpression of GFP-tagged flotillin 1 and flotillin 2 in HBMEC inhibited E. coli Kl invasion, which suggest flotillin might have a role as a regulatory cell barrier in host defence.

Blastocystis is an obligate anaerobic, protistan parasite found in the intestinal tract of human and various other hosts. Blastocystis is placed within the stramenopiles. This diverse group also includes slime nets, water moulds and brown algae. The transmission of Blastocystis is believed to take place through the faecal-oral route. Waterborne transmission of Blastocystis through the use of untreated water or poor sanitary conditions has also been reported. Numerous epidemiological surveys have shown prevalence up to 10% of the population in developed countries and as much as 50-60% in developing countries. Differences in virulence among Blastocystis subtypes have been reported in a recent animal infection study. Blastocystis shows extensive genetic diversity and is divided into numerous genetic subtypes The parasite is commonly associated with gastrointestinal symptoms such as watery and mucous diarrhea, vomiting, and abdominal cramps and bloating. Epidemiological studies suggest an association between Blastocystis infection and irritable bowel syndrome. Irritable bowel syndrome (IBS) is identified as a functional bowel disorder in which abdominal pain or discomfort is associated with a defect or alteration in the consistency or frequency of stools. Diagnosis of IBS by physicians is carried out using symptom -based criteria known as the Rome criteria. To see whether there is any link between Blastocystis infection and irritable bowel syndrome (IBS), I have compared the frequency of subtypes of Blastocystis in IBS patients with those in the general population. In human population both UK and Libya showed similar distribution of Blastocystis subtypes 2 ST I, 2 and 3 are common in the two populations and ST3 has the highest frequency in UK while STI was the most common in Libya. Epidemiology studies on Blastocystis infection in animals have revealed high frequency of occurrence in cattle, pigs, primates and birds and it has often been suggested that Blastocystis infection is a zoonosis. In Libya, Blastocystis subtypes were detected from humans, birds and numerous mammals' hosts (camel, cow, sheep, goat, gazelle, Barbary sheep and gundi). Ten subtypes were detected (1, 2, 3, 5, 7,10,14, IS, 16, 17) and four new subtypes were found in cow, camel and gundi. Subtype I, 3, and 7 were in common between animal and human but subtypes 5, 10 and the four new subtypes were found only in animals. ST 2 was found in human only. Also I discovered four new hosts for Blastocystis from mouse deer, gundi, gazelle and barbary sheep. Both human and animal showed diversity in Blastocystis sUbtypes. Both human and animals become infected with same Blastocystis subtype and for this reason we need to find refined tool to differentiate between them, so I have developed MLST for Blastocystis subtype I based on mitochondrial DNA. Application ofMLST to 39 isolates from different host and different geographic place showed variation in the sequence of related isolates. Over all MLST proved to be a highly discriminatory and stable method for unambiguous characterization of Blastocystis.

Anthroponotic cutaneous leishmaniasis caused by Leishmania tropica has been endemic in Aleppo, Syria for centuries. The first modem description of the disease was also done in Aleppo. A surveillance system is in place, and the numbers of annual recorded cases have been rising from a few hundred to thousands in the late 1980s, to more than 5,000 in most years from 1990, and to more than 10,000 since 2003. A retrospective analysis of routinely collected demographic data was performed. The clinical course was examined in a subset of patients. One hundred and thirty-two patients were recruited for follow-up study. Parasites were isolated from the lesions of these patients before treatment and during the course of treatment. Eighty isolates were tested for drug sensitivity in amastigotemacrophage system and typed to species level. Molecular fingerprinting was applied to a subset of isolates. Interviews were held with patients or accompanying adults about their knowledge, attitudes and practices regarding prevention, diagnosis and treatment. Leishmaniasis patients in Aleppo were younger than the general population (median age 13 vs. 19 years), and females predominated among adults. Children and males were more likely to have lesions on the face. Smear positivity decreased with patient age (OR=O.5 in over-forties compared to under-tens). Smear positivity peaked at two-month lesion duration (OR=2.2 compared to lesion duration of <1 month). A significant proportion of patients, especially adults, did not complete their treatment course. The isolated parasites were insensitive (median ECso=229 fig Sbv Iml) to pentavalent antimony, the drug used in Aleppo, and to paromomycin but were sensitive to amphotericin B. No relationship was found between baseline parasite in vitro sensitivity and treatment duration. All the typed parasites were L. tropica. Parasite schizodemes clustered by place of isolation and by family.

Malaria is widely perceived as immunosuppressive. Despite extensive phenomenological description, the underlying mechanisms remain poorly described. The aim of this thesis was to identify possible mechanisms by which malaria modifies host defence, and to determine the importance of these mechanisms in a translational system moving from a mouse model to human malaria. The most frequently cited immunological consequences of malaria are: suppression of vaccine responses, susceptibility to bacterial infection, susceptibility to endemic Burkitt lymphoma, and increased HIV viral load. Of these, susceptibility to non-Typhoid Salmonella (NTS) bacteremia, associated with severe hemolysis, was the most consistent between animal and human studies. I hypothesized that hemolysis would induce the immunomodulatory enzyme heme oxygenase-l (HO-l), which is essential for survival in malaria infections in mice, but might impair host defence against NTS. I demonstrate in mice that malaria, chemically-induced hemolysis, or simply administration of heme, cause loss of resistance to NTS, allowing more rapid bacterial growth than in control animals. A new niche for bacterial replication is established within neutrophils, which have impaired oxidative burst and bacterial killing activity. Hemolysis and heme induce HO-1 in neutrophil progenitors in the bone marrow, and this reduces the oxidative burst capacity of maturing neutrophils whilst also causing their premature mobilization into the circulation. Inhibition of HO by the competitive inhibitor SnPP abrogates the impaired resistance to NTS infection. I observed a similar phenomenon in Gambian children with malaria, with prolonged impairment of neutrophil function, the severity of which is related to hemolysis and HO-l induction. In summary I have shown that hemolysis- and HO-l-mediated neutrophil dysfunction occurs in malaria and is important for susceptibility to NTS infection. HO-1 inhibition might offer a novel therapy to alleviate neutrophil dysfunction in malaria patients.

Plasmodium knowlesi infections in humans have been increasingly seen in many countries across Southeast Asia, with cases mainly concentrated in Malaysia, since a major focus of infections was first described in Malaysian Borneo over 10 years ago. Clinical presentations show a wide spectrum of illness from mild to fatal, with the possible occurrence of asymptomatic infections. Two monkey species have been identified as the chief reservoir hosts; long-tailed macaque (Macaca fascicularis) and pig-tailed macaque (M. nemestrina). In order to explore the transmission of P. knowlesi infections, it is important to study the population genetic structure of this parasite. To address this, a microsatellite genotyping toolkit consisting of 10 loci specific for P. knowlesi was developed and validated. Using these highly polymorphic markers, analysis of more than 500 P. knowlesi infections from human and wild macaque hosts across Malaysian Borneo and humans of peninsular Malaysia showed remarkable population genetic structure. Human clinical isolates were shown to comprise highly divergent subpopulations, respectively associated with forest-dwelling long-tailed macaque (Cluster 1) and pig-tailed macaque (Cluster 2) reservoir hosts. After analysis of initial whole genome sequence data, re-assessment of population genetic structure was undertaken by microsatellite analysis of more samples from wild macaques and humans from peninsular Malaysia, showing profound geographical divergence between Borneo (sympatric Cluster 1 and Cluster 2) and mainland peninsular Malaysia (Cluster 3). The overall three major subpopulations demonstrated by microsatellite 4 analyses matched the analysis inferred by the genome-wide sequence analysis of clinical isolates. To allow further investigation of variation in genome-wide divergence between the sympatric subpopulations in Borneo, a simple laboratory kit consisting of allele-specific PCR assays was developed to distinguish the two subpopulations. This eased in identifying to which subpopulations P. knowlesi infections belonged, and subsequently generating more genome-wide sequences for comparative study. Further analyses revealed remarkable heterogeneity in the level of divergence between the sympatric subpopulation across the genome. Genomic architectures showed 20 high-divergence regions scattered in different chromosomes. These findings suggest independent adaption of parasites in different macaque hosts that persist sympatrically.

Tuberculosis and helminth infections are co endemic in many parts of the world. This geographical overlap has led to the hypothesis that helminth infections could exacerbate the effects of Mycobacterium tuberculosis (Mtb) infection. Anthelmintic treatment has been observed to be associated with improved mycobacterial cellular responses and decreases in the frequency of Treg cells. The consequence of this immunomodulation may affect the ability of the host to restrict the growth of mycobacteria or mycobacterial killing. This study aims at investigating the modulations of the immune response profile of latent tuberculosis (LTBI) and helminth co-infected patients and whether these modulations are associated with a decrease in mycobacterial growth inhibition using a mycobacterial growth inhibition assay (MGIA).UK migrants attending University College Hospital London, UK with eosinophilia or suspected/diagnosed helminth infection (Strongyloides spp and Schistosoma spp) and/or LTBI were bled at recruitment (before anthelmintic treatment) and 4 months after completing anthelmintic treatment. Helminth infected patients displayed poor growth inhibition on MGIA which was improved after anthelmintic treatment which indicated this immunomodulation might be helminth mediated. The percentage of CD4+ T cells expressing IFNg, TNFa and IL-2 were quantified by flow cytometry in PPD and ESAT-6/CFP-10 stimulated PBMC and anthelmintic treatment was observed to increase the frequency of CD4+IFNg response. LTBI-helminth coinfection was associated with significantly elevated levels of proinflammatory and lower levels of anti-inflammatory cytokines after they were treated. IP- 10 was significantly upregulated and MCP-1 was significantly downregulated in LTBIhelminth coinfected patients after anthelmintic treatment. The effect of IL-10 and TGFb on MGIA were observed and suggested an immunoregulatory role in helminth infected patients. Gene expression analysis by qRT-PCR showed varied responses and showed significant fold changes of CXCL-10, arginase 1 and CD163 after the treatment. MGIA and multiple immune parameters have shown that helminth infection can modulate a variety of Mtb specific immune responses.

Toxoplasma gondii is a globally distributed protozoan parasite that infects humans and a wide variety of warm-blooded animals. Although there are many surveys for T. gondii infection in mammals, little is known about the detailed distribution in localised natural populations. In this study we investigated host genotype and spatial location in relation to T. gondii infection and genotypes. We collected wood mice (Apodemus sylvaticus) from 4 sampling sites within a localised peri-aquatic woodland ecosystem which is relatively free of cats. Mice were genotyped using standard A. sylvaticus microsatellite markers and T. gondii and its genotypes were detected using 5 specific PCR based markers: SAG1, SAG2, SAG3, B1 and GRA6 directly from infected tissue. Of 126 wood mice collected, 44 samples gave positive reactions with T. gondii specific markers giving an infection rate of 34.92% (95% CI: 27.14%-43.59%). A total of 24/76 (31.58%, 95% CI: 22.19%-42.74%) males and 20/50 (40%, 95% CI: 27.59%-53.84%) female mice were found to be positive for T. gondii with no significant difference (P = 0.353). Juvenile, young adults and adults were infected at similar prevalences, respectively, 7/17 (41.18%), 27/65 (41.54%) and 10/44 (22.72%) with no significant age-prevalence effect (P = 0.23). Detailed analysis of the RFLP patterns and the DNA sequences for the SAG2, SAG3 and GRA6 loci showed a range of genotypes but, surprisingly, suggested that 30/44 (68.2%) infected mice had multiple genotypes (mixed infections) present. Results of genetic analysis of the mice showed that the collection consists of four genetically distinct populations. There was a significant difference in T. gondii infection in the different mouse genotypically derived populations (P=0.035) but not between geographically defined populations based on sampling location (P=0.29). In a parallel study, DNA was successfully collected from 88 human lung tissue samples. All samples showed successful amplification of the human α-tubulin gene and were used for T. gondii DNA detection. We used commonly used PCR markers (B1, SAG1, SAG2, SAG3, GRA6, APICO, L358, PK1, SAG1-Su, BTUB, alt.SAG2, c22-8 and c29-2), histological and immunohistochemical staining to confirm the presence of the parasite. All 88 tested samples were confirmed to be positive for T. gondii with markers B1, SAG1, SAG2 3’, SAG2 5’ and SAG3, giving a prevalence of 100% (95% CI: 95.82%-100%). From all successfully genotyped samples, 34 had single infection on all loci and 42 were of mixed infection on one or more loci with all three genotypes present. Type II genotype was the most predominant, followed by Type I and Type III. We detected 11 unusual genotypes. Immunohistochemistry was performed on 76 of the 88 tissue sections using commercial polyclonal antibodies produced in rabbits. All 76 sections were confirmed to be positive for T. gondii. A surprisingly high number of patients (96.05%) showed evidence of an active form of infection, as defined by the presence of tachyzoites or infected alveolar macrophages (or other cell types). Only three subjects (3.95%) had the dormant cyst stage as the only stage present. All 76 tissue sections were successfully stained with haematoxylin and eosin and observed under the light microscope. The presence of structures consistent with infection by the parasite was confirmed in 67 samples. All these patients are at risk of reactivation of chronic infection, leading to toxoplasmic encephalitis or pulmonary toxoplasmosis; which can complicate and delay their treatment or lead to death.

No description available.

The bacterial cell envelope heteropolymer, peptidoglycan (PG), is essential for maintaining the osmotic stability and shape of most bacteria. PG biosynthesis is the target of our most successful antibiotics, the β-lactams and glycopeptides. However, the spread of antibiotic resistant strains highlights the need for novel antibiotic targets. Gram-negative bacteria possess a mainly single layered PG, which is enlarged in growing and dividing bacteria by the coordinated action of PG synthases and hydrolases. PG synthesis in Gram-negative bacteria is regulated from the cytoplasmic membrane (CM), by prokaryotic cytoskeletal elements, and from the outer membrane (OM) by the lipoproteins, LpoA and LpoB. LpoA/B interact with, and are essential for the in vivo activity of, the major PG synthases PBP1A and PBP1B, respectively. While the regulation of PG synthesis has been well studied in recent years, the mechanisms of PG hydrolysis regulation in E. coli remain poorly understood. E. coli possesses ~30 PG hydrolases with relatively few known regulators. In this work, we have structurally characterised LpoA from E. coli using nuclear magnetic resonance (NMR) spectroscopy of the N-terminal domain and use this to further the understanding of the in vitro and in vivo interaction of LpoA/PBP1A. We also studied PBP1A and LpoA in Haemophilus influenzae; in this species LpoA is essential. In a search for novel LpoA interaction partners we discovered the in vitro and in vivo interaction with the PG hydrolase, PBP4 and show that PBP4 also interacts with PBP1A. Subsequently, we optimised a process for the rapid identification of in vitro interactions and identified >20 interactions between PG synthases, PG hydrolases and other cell envelope proteins. We therefore present a putative PG hydrolysing complex with direct associations to the PG synthesis machinery. Through direct functional interactions with at least five PG hydrolases, we present the characterisation of the OM-anchored lipoprotein NlpI, of currently unknown cellular function, as a regulator of hydrolase activity. We show the in vitro regulation of activity by NlpI and the in vivo relevance of these interactions using a β-lactamase induction assay. This work significantly enhances our understanding of how PG synthesis and hydrolysis are coordinated as multi-enzyme complexes and presents the characterisation of a novel regulator of hydrolase activity, NlpI.

The pathogenesis of Campylobacter enteritis is not well understood including the mechanisms involved in invasion and translocation across intestinal epithelial cells. The genetic make-up of the pathogen and its responses to different environmental cues are thought to contribute to the organism's ability to survive and cause disease. The extremes of environment which Campylobacter can with-stand, and the effect that this has on virulence and invasive ability remains undefined. For the first time, several isolates were compared quantitatively to determine the extent to which intracellular invasion contributes to translocation across epithelial cell mono layers. Translocation ability did not correlate with intracellular invasiveness, suggesting that different "invasion" phenotypes exist among Campylobacler isolates. Repeated exposure of Campylobacler isolates to Caco-2 cells caused an increase in their ability to invade and survive, which was associated with changes in protein expression. Campylobacler was grown in continuous culture under conditions of iron sufficiency, iron limitation, oxidative stress and low pH. Uniquely, growth under oxidative stress and iron replete conditions caused an increase in the invasive ability of C. jejuni 81116, which was correlated with the up-regulation of specific proteins. The role of three proteins, HtrB, Tpx and PEB-4, was investigated at the molecular level. Two of the encoding genes, peb4A and hlrB, were found to be essential for viability. Homologous recombination of an inactivated Ipx gene into the genome of C. jejuni caused increased sensitivity to H20 2, but did not affect the ability of C. jejuni to invade and survive within Caco-2 cell monolayers. This study demonstrated that isolates of Campylobacter differ significantly in their virulence potential with respect to their invasive phenotypes. In addition Campylobacler grown in well defined continuous culture conditions demonstrated for the first time the importance of iron and oxidative stress as acting as potenital cues for the expression of survival and invasion determinants.