Browsing by Author "Spagnuolo, Julian"

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    Defining the gate domain of the filamentous phage secretin pIV
    (Massey University, 2010) Spagnuolo, Julian
    Secretins are a family of large outer membrane proteins with large-diameter lumens (5-10 nm). This allows them to transport bulky substrates, including folded proteins, or assembled macromolecular structures – filamentous phages and type IV pili. Many proteins exported by secretins are essential for virulence of Gram-negative pathogens. Such a large channel would ordinarily sensitize the bacterial cell to noxious agents. However, secretins do not - the presence of a mobile septum, or gate, across the lumen of the channel prevents access by noxious agents. Despite the importance of the gate in secretin function, the sequence identity of the gate residues is unknown. In this study, in vivo random mutagenesis was used to map amino acid residues involved in gating the filamentous phage secretin - pIV. This approach has identified 34 residues that are involved in the gating mechanism. These residues are predominantly located within the secretin homology domain and organised into two clusters; GATE1 (39 residues) and GATE2 (14 residues). A number of isolated point mutants sensitised Escherichia coli to bile salts and antibiotics. These findings allowed the construction of a site-directed deletion mutant of GATE2, confirming the gate function. This thesis mapped, for the first time, a secretin gate. Given the success of the mutagenesis approach used in this thesis, the method here will be applicable to secretins of pathogenic bacteria and other outer membrane channels whose gate regions have not been determined as yet. Knowing the gate regions of “pathogenic” secretins in turn will help design secretin-targeting antimicrobials.
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    Extracytoplasmic stress responses induced by a model secretin : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2015) Spagnuolo, Julian
    Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated transenvelope multiprotein machinery, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria, type 2/3 secretion systems, filamentous phage assembly-secretion system and the type 4 pilus assembly system contain large homologous gated channels, called secretins, in the outer membrane. Secretins are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum or gate. Expression of these channels imposes a fitness cost to bacteria. While stress induced by model secretin pIV has been previously investigated using microarrays, this thesis is the first RNAseq characterisation of secretin stress responses. Furthermore, this is the first comparison of stress imposed by a closed-gate secretin (wildtype pIV), vs. an isogenic leaky-gate variant, the latter serving as a model of an open-gate substrate-secreting channel. The high sensitivity to changes in gene expression and low background noise of the RNA-seq approach have greatly expanded the known secretin stress responses to include the SoxS, CpxR and RcsB/RcsAB regulons, in addition to the known involvement of the Psp response. A synthetic lethality analysis of candidate genes in these pathways suggested that the leaky-gate secretins, besides rendering the Psp response essential for survival, also stimulate the SoxS and RcsB/RcsAB regulons for protection of the cells. Knowledge of the secretin stress expanded by this work helped identify potential targets for development of much-needed antibiotics against toxinsecreting Gram-negative bacteria.