Impairment of cell adhesion and migration by inhibition of protein disulphide isomerases in three breast cancer cell lines.

Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified 'scratch wound' assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3-/- mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3-/- MEFs was less effective in promoting cell spreading and F-actin organisation or supporting 'scratch wound' closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

O-mannosylation in Candida albicans enables development of interkingdom biofilm communities.

Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. Formation of dual-species S. gordonii-C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans. Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1Δ mnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii. Cell wall proteomes of hypha-forming mnt1Δ mnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1Δ mnt2Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1Δ mnt2Δ mutant. These observations imply that early stage O-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. IMPORTANCE In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus. We find that impairment of early stage addition of mannose sugars to C. albicans hyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to control C. albicans infections.

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression.

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE(+) and SopE(-) S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH-gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA-gfp reporter mirrored that of PprgH-gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.