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Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

ABSTRACT: Staphylococcus aureus is a notorious human pathogen that relies on an array of virulence factors to engender infection and evade the host-immune system. Among these are the secreted proteases, which promote pathogenesis by degrading host proteins and modulating host-defenses. Human neutrophils play a pivotal role in these defenses, acting as the first responders against invading bacteria. While many S. aureus effectors of virulence have been shown to target leukocytes, there is limited knowledge on how the extracellular proteases modulate neutrophil fate. Typically, protease substrates have been identified in isolated settings using one at a time approaches; with neutrophil targets few and far between. Herein, we have developed a novel N-terminomic methodology termed TAGS-CR that can facilitate global substrate characterization in streamlined manner. We thus present the application of TAGS-CR to unravelling the human neutrophil pathodegradome of the S. aureus V8 protease. In so doing, we captured ~350 V8 targets, revealing critical insight into how this virulence factor can modulate neutrophil functionality on various levels relevant to S. aureus disease progression. We recorded cleavage of proteins necessary for neutrophil adhesion and migration, a fundamental process necessary for pathogen clearance. Furthermore, we highlight V8 cleavage of proteins involved in important neutrophil defense tactics, such as degranulation and reactive oxygen species production. This protease may also facilitate bacterial dissemination via the intentional activation of neutrophil apoptosis. Collectively, this work deepens our understanding of host-pathogen interaction and begins to unravel how S. aureus proteases can induce immune dysregulation through the targeting of leukocytes.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Emilee Mustor

LAB HEAD: Lindsey N. Shaw

PROVIDER: PXD057579 | Pride | 2025-05-30

REPOSITORIES: Pride

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			Action	DRS
	20240712_BAK_gel_lane4.raw	Raw
	20240712_BAK_gel_lane5.raw	Raw
	20240712_BAK_gel_lane6.raw	Raw
	mqpar_BAK1_GelDigest.xml	Xml
	txt_BAK1_GelDigest.zip	Other

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Project description:This study was designed to identify changes in gene expression when corn was placed under various related stresses including being grown with a competing weed (canola) to the V4 or V8 stage, or when 40% shade cloth was present to the V4 or V8 stage, or under low nitrogen (no added nitrogen fertilizer), or under weed/shade free fertilized control conditions. In all 5 treatments and the control, samples were harvested at V8. Mechanisms underlying early season weed stress on crop growth are not well described. Corn vegetative growth and development, yield, and gene expression response to nitrogen (N), light (40% shade), and weed stresses were compared with the response of nonstressed plants. Vegetative parameters, including leaf area and biomass, were measured from V2 toV12 corn stages. Transcriptome (2008) or quantitative Polymerase Chain Reaction (q PCR) (2008/09) analyses examined differential gene expression in stressed versus nonstressed corn at V8. Vegetative parameters were impacted minimally by N stress although grain yield was 40% lower. Shade, present until V2, reduced biomass and leaf area > 50% at V2 and, at V12, recovering plants remained smaller than nonstressed plants. Grain yields of shade-stressed plants were similar to nonstressed controls, unless shade remained until V8. Growth and yield reductions due to weed stress in 2008 were observed when weeds remained until V6. In 2009, weed stress at V2 reduced vegetative growth, and weed stress until V4 or later reduced yield. Principle component analysis of differentially expressed genes indicated that shade and weed stress had more similar gene expression patterns to each other than to nonstressed or low N stressed tissues. Weed-stressed corn had 630 differentially expressed genes compared with the nonstressed control. Of these genes, 259 differed and 82 were shared with shade-stressed plants. Corn grown in N-stressed conditions shared 252 differentially expressed genes with weed-stressed plants. Ontologies associated with light/photosynthesis, energy conversion, and signaling were down-regulated in response to all three stresses. Although shade and weed stress clustered most tightly together, only three ontologies were shared by these stresses, O-methyltransferase activity (lignification processes), Poly U binding activity (post-transcriptional gene regulation), and stomatal movement. Based on both morphologic and genomic observations, results suggest that shade, N, and weed stresses to corn are regulated by both different and overlapping mechanisms. three biological replicates for each treatment and the control were collected and the resulting labeled cDNA was hybridized to the 46,000-element maize microarray chip developed by the University of Arizona using their protocol (International Microarray Workshop Handbook, 2009Gardiner et al. 2005). The hybridization scheme was a dual hybridization using a rolling circle balanced dye swap design. Thus we had thre biological replicates for each growth condition amd two technical replicates for each biological sample.

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Development of a New N-Terminomic Method to Study the Pathodegradome of the Staphylococcus aureus V8 Protease in Human Neutrophils

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