Characterisation of proteins extracted from the surface of Salmonella Typhimurium grown under SPI‐2‐inducing conditions by LC‐ESI/MS/MS sequencing

Abstract

Salmonella enterica has two pathogenicity islands encoding separate type three secretion systems (T3SS). Proteins secreted through these systems facilitate invasion and survival. After entry, Salmonella reside within a membrane bound vacuole, the Salmonella containing vacuole (SCV), where translocation of a second set of effectors by the Salmonella pathogenicity island 2 (SPI-2) T3SS is initiated. SPI-2 secretion in vitro can be induced by conditions that mimic the Salmonella containing vacuole. Utilising high-throughput mass spectrometry, we mapped the surface-attached proteome of S. Typhimurium SL1344 grown in vitro under SPI-2-inducing conditions and identified 108 proteins; using secretion signal prediction software, 43% of proteins identified contained a signal sequence. Of these proteins, 13 were known secreted effector proteins including SPI-2 effector proteins SseB, SseC, SseD, SseL, PipB2 and SteC, although surprisingly five were SPI-1 proteins, SipA, SipB, SipC, SipD and SopD, while 2 proteins SteA and SlrP are secreted by both T3SSs. This is the first in vitro study to demonstrate dual secretion of SPI-1 and SPI-2 proteins by S. Typhimurium and demonstrates the potential of high-throughput LC-ESI/MS/MS sequencing for the identification of novel proteins, providing a platform for subsequent comparative proteomic analysis, which should greatly assist understanding of the pathogenesis and inherent variation between serovars of Salmonella and ultimately help towards development of novel control strategies.

Flagellin Is Required for Host Cell Invasion and Normal Salmonella Pathogenicity Island 1 Expression by Salmonella enterica Serovar Paratyphi A

“…Similar results were also obtained for many of the secreted flagellar components (Table 2). Other peptidoglycan-associated lipoproteins, such as DnaK and GroEL (27), were identified in similar amounts, suggesting that the increased secretion is specific to the exported flagellar and SPI-1 proteins.…”

Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors

“…Nevertheless, most SPI-2 T3SS effectors do require the two-component regulatory system SsrA/B for their expression, and genetic screens exploiting this dependency have identified several additional effectors (Coombes et al, 2007;Rytkönen et al, 2007;Worley et al, 2000). Other methods include the use of CyaA transposon fusions to identify translocated proteins (Geddes et al, 2005), and recent studies have taken advantage of the phenotype of an ssaL mutant strain (which is derepressed for effector secretion; Yu et al, 2010) and conditions that induce SPI-2 gene expression, to identify known and new effectors by MS analysis of proteins in bacterial culture supernatants (Auweter et al, 2011;Niemann et al, 2011;Sherry et al, 2011). Together, these and other approaches have identified 28 effectors of the SPI-2 T3SS (Table 1, Fig.…”

Proteomic Characterisation of Two Strains of Mycoplasma mycoides Subsp. Mycoides of Differing Pathogenicity

“…These procedures were carried out on both Mycoplasma whole cells and on Triton-X 114 membrane enriched fraction as described previously in similar surveys of bacterial proteomes [21][22][23][24][25] summarized as follows:…”