Characterisation of adhesion of a probiotic bacterium Lactobacillus rhamnosus HN001 to extracellular matrix proteins and the intestinal cell line Caco-2 : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Master of Science in Microbiology

This study focuses on Lactobacillus rhamnosus HN001, a potential candidate for use as a probiotic. Probiotics are microorganisms that can exert a beneficial effect on a host. It is believed that the ability of a probiotic to colonise gastrointestinal surfaces is important in its ability to exert a beneficial effect on the host. In order to do so, it is thought the microorganism must be able to adhere to molecules found on intestinal cells. HN001 has been shown to adhere to human intestinal cell lines (Gopal et al., 2001). This study characterises the molecular species involved in the adherence of HN001 to intestinal molecules and cell lines, which may be important in the ability of HN001 to exert health benefits in a host. Both liquid and solid-phase binding assays were used to characterise HN001 binding to extracellular matrix (ECM) components found in intestinal tissues. Of the ECM components investigated, HN001 bound fibronectin with the highest affinity. This interaction was specific, saturable and dependent on the growth phase of HN001. HN001 bound immobilised fibronectin in preference to soluble fibronectin through a protein-dependent interaction. HN001 was also found to bind to the N-terminal heparin binding domain of fibronectin and the C-terminal part of the first type III repeat in the fibronectin molecule (III1-C). HN001 adhered to the human intestinal cell line, Caco-2, in a dose-dependent manner that was enhanced by a pH-sensitivc factor present in the spent culture supernatant. Since fibronectin-binding was identified as a possible mechanism for adherence of HN001 to intestinal tissues, HN001 genome DNA sequence was examined for genes encoding putative fibronectin-binding proteins. Fbl (Fibronectin-binding like) was identified through its similarity to fibronectin-binding proteins from Streptococcus pneumoniae (Holmes et al., 2001) and S. pyogenes (Courtney et al., 1994). Fbl was expressed by a GST fusion system and used to compete with HN001 adhesion in liquid-phase binding assays to ascertain its function. Since difficulties were experienced when expressing and purifying soluble Fbl, an insertional disruption of the fbl gene was created and its phenotype investigated in liquid-phase, solid-phase and Caco-2 binding assays to determine Fbl function

Sequence Comparisons of Odorant Receptors among Tortricid Moths Reveal Different Rates of Molecular Evolution among Family Members

In insects, odorant receptors detect volatile cues involved in behaviours such as mate recognition, food location and oviposition. We have investigated the evolution of three odorant receptors from five species within the moth genera Ctenopseustis and Planotrotrix, family Tortricidae, which fall into distinct clades within the odorant receptor multigene family. One receptor is the orthologue of the co-receptor Or83b, now known as Orco (OR2), and encodes the obligate ion channel subunit of the receptor complex. In comparison, the other two receptors, OR1 and OR3, are ligand-binding receptor subunits, activated by volatile compounds produced by plants - methyl salicylate and citral, respectively. Rates of sequence evolution at non-synonymous sites were significantly higher in OR1 compared with OR2 and OR3. Within the dataset OR1 contains 109 variable amino acid positions that are distributed evenly across the entire protein including transmembrane helices, loop regions and termini, while OR2 and OR3 contain 18 and 16 variable sites, respectively. OR2 shows a high level of amino acid conservation as expected due to its essential role in odour detection; however we found unexpected differences in the rate of evolution between two ligand-binding odorant receptors, OR1 and OR3. OR3 shows high sequence conservation suggestive of a conserved role in odour reception, whereas the higher rate of evolution observed in OR1, particularly at non-synonymous sites, may be suggestive of relaxed constraint, perhaps associated with the loss of an ancestral role in sex pheromone reception

FUS pathology defines the majority of tau- and TDP-43-negative frontotemporal lobar degeneration

Through an international consortium, we have collected 37 tau- and TAR DNA-binding protein 43 (TDP-43)-negative frontotemporal lobar degeneration (FTLD) cases, and present here the first comprehensive analysis of these cases in terms of neuropathology, genetics, demographics and clinical data. 92% (34/37) had fused in sarcoma (FUS) protein pathology, indicating that FTLD-FUS is an important FTLD subtype. This FTLD-FUS collection specifically focussed on aFTLD-U cases, one of three recently defined subtypes of FTLD-FUS. The aFTLD-U subtype of FTLD-FUS is characterised clinically by behavioural variant frontotemporal dementia (bvFTD) and has a particularly young age of onset with a mean of 41 years. Further, this subtype had a high prevalence of psychotic symptoms (36% of cases) and low prevalence of motor symptoms (3% of cases). We did not find FUS mutations in any aFTLD-U case. To date, the only subtype of cases reported to have ubiquitin-positive but tau-, TDP-43- and FUS-negative pathology, termed FTLD-UPS, is the result of charged multivesicular body protein 2B gene (CHMP2B) mutation. We identified three FTLD-UPS cases, which are negative for CHMP2B mutation, suggesting that the full complement of FTLD pathologies is yet to be elucidated

Likelihood ratio tests between concatenated sequences.

<p>model A assumes complete homogeneity among genes, while model B assumes different substitution rates but the same pattern of nucleotide substitution for each gene.</p

Summary statistics for odorant receptors OR1, OR2 and OR3 from <i>Ctenopseustis</i> and <i>Planotortrix</i> species.

a<p>number of sequences.</p>b<p>number of codons.</p>c<p>tree length.</p>d<p>transition/transversion ratio.</p>e<p>dN/dS under M0.</p>f<p>dN/dS under M3.</p

Unrooted phylogenetic tree of all lepidopteran odorant receptors within Genbank as of 8 November, 2010.

<p>The neighbour joining tree was constructed from dayhoff amino acid distances. The positions of OR1, OR2 (Orco), and OR3 from <i>Epiphyas postvittana</i> are indicated with arrows (EpOR1, EpOR2, and EpOR3), while the Orco and sex pheromone receptor clades are highlighted by semicircles.</p

Amino acid identity matrix for <i>Ctenopseustis</i> and <i>Planotortrix</i> orthologues of OR1, OR2 and OR2.

a<p>Ctenopseustis obliquana.</p>b<p>C. herana.</p>c<p>Planotortrix excessana.</p>d<p>P. octo.</p>e<p>P. notophaea.</p>f<p>Epiphyas postvittana.</p>g<p>BmOR1.</p>h<p>BmOR2.</p>i<p>BmOR49.</p

Ratio of the relative amino acid differences per domain averaged for OR1, OR2 (Orco) and OR3 across <i>Ctenopseustis obliquana</i>, <i>C. herana</i>, <i>Planotortrix octo</i>, <i>P. excessana</i> and <i>P. notophaea</i>.

<p>The ratio for each domain is the average of the number of amino acid differences divided by the number of expected differences. Expected differences were calculated by multiplying the length of the domain by the total number of differences per protein then dividing by the length of the protein. The ratio would be 1 if the amino acid changes occurred at the same rate across the entire protein. N-ter = N terminus; TM1-TM7 = transmembrane domains 1–7; IL1-3 = internal loops 1–3; EL1-3 = external loops 1–3; C-ter = C terminus. ND = not determined.</p

Predicted transmembrane topologies of OR1 (A), OR2 (B) and OR3 (C), with variable sites highlighted in red.

<p>The double line indicates the membrane region, with extracellular and cytoplasmic sides labelled.</p