Comparative Proteomic Analysis of Adhesion/Invasion Related Proteins in Cronobacter sakazakii Based on Data-Independent Acquisition Coupled With LC-MS/MS.

Ping Li,Shuo Wang,Xuan Dong,Xiao-Yi Wang,Xin-Jun Du,Ting Du

doi:10.3389/fmicb.2020.01239

Ping Li, Shuo Wang + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2020.01239

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Abstract

Cronobacter sakazakii is foodborne pathogen that causes serious illnesses such as necrotizing enterocolitis, meningitis and septicemia in infants. However, the virulence determinants and mechanisms of pathogenicity of these species remain unclear. In this study, multilocus sequence typing (MLST) was performed on 34 C. sakazakii strains and two strains with the same sequence type (ST) but distinct adhesion/invasion capabilities were selected for identification of differentially expressed proteins using data-independent acquisition (DIA) proteomic analysis. A total of 2,203 proteins were identified and quantified. Among these proteins, 210 exhibited differential expression patterns with abundance ratios ≥3 or ≤0.33 and P values ≤0.05. Among these 210 proteins, 67 were expressed higher, and 143 were expressed lower in C. sakazakii SAKA80220 (strongly adhesive/invasive strain) compared with C. sakazakii SAKA80221 (weakly adhesive/invasive strain). Based on a detailed analysis of the differentially expressed proteins, the highly expressed genes involved in flagellar assembly, lipopolysaccharide synthesis, LuxS/AI-2, energy metabolic pathways and iron-sulfur cluster may be associated with the adhesion/invasion capability of C. sakazakii. To verify the accuracy of the proteomic results, real-time qPCR was used to analyze the expression patterns of some genes at the transcriptional level, and consistent results were observed. This study, for the first time, used DIA proteomic to investigate potential adhesion/invasion related factors as a useful reference for further studies on the pathogenic mechanism of C. sakazakii.

Highlights

Cronobacter species are rod-shaped, peritrichous, motile, Gram-negative, non-spore-forming facultative anaerobes that are widely present in nature (Iversen and Forsythe, 2003)
This study is the first time to investigate proteomic profiles of C. sakazakii using LC-MS/MS coupled with data-independent acquisition (DIA) labeling
Based on our previous results (Du et al, 2016) from adhesion/invasion analysis coupled with sequence typing, we selected the strong-adhesive/invasive strain SAKA80220 and the weak-adhesive/invasive strain SAKA80221 as strains that exhibited distinct adhesion/invasion capabilities but were close in evolutionary relationship for comparative proteomics analysis

Summary

Introduction

Cronobacter species are rod-shaped, peritrichous, motile, Gram-negative, non-spore-forming facultative anaerobes that are widely present in nature (Iversen and Forsythe, 2003). Proteomic Analysis of C. sakazakii are associated with severe infections (Forsythe, 2018). Several studies using gene knockouts have demonstrated that outer membrane protein A (OmpA), outer membrane protein X (OmpX) and outer membrane protease Cpa as well as the gene bcsR are crucial virulence factors of Cronobacter (Nair et al, 2009; Kim et al, 2010; Franco et al, 2011; Singh et al, 2015; Gao et al, 2017). It has been demonstrated that enterotoxins, lipopolysaccharides (LPSs) and flagella are closely associated with the virulence of Cronobacter (Hotta et al, 1986; Pagotto et al, 2003; Jing et al, 2016). Further studies on the pathogenesis of Cronobacter are necessary to understand these bacteria and to establish effective strategies for prevention and therapy

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