Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis

    Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis Yingwang Ye, Hui Li, Na Ling, Yongjia Han, Qingping Wu, Xiaoke Xu, Rui Jiao, Jina Gao PII: DOI: Reference:

S0168-1605(15)30112-4 doi: 10.1016/j.ijfoodmicro.2015.08.025 FOOD 7030

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

21 June 2015 24 August 2015 30 August 2015

Please cite this article as: Ye, Yingwang, Li, Hui, Ling, Na, Han, Yongjia, Wu, Qingping, Xu, Xiaoke, Jiao, Rui, Gao, Jina, Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.08.025

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ACCEPTED MANUSCRIPT Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis ＃

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School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009,

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Yingwang Ye1,2 , Hui Li1 , Na Ling1,2 , Yongjia Han1 , Qingping Wu2 , Xiaoke Xu2, Rui Jiao1, Jina Gao1

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State Key Laboratory of Applied Microbiology, South China (the Ministry–Province Joint

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Development), Guangdong Provincial Key Laboratory of Microbiology Culture Collection and

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Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China : Corresponding author: Xianlie Central Road 100, Guangzhou city, Guangdong Province, China,

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: Authors contribute to the manuscript equally

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510070. E-mail: [email protected] (Qingping Wu). Tel: 86-20-87688132

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ACCEPTED MANUSCRIPT Abstract: Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter

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sakazakii is the most common species in terms of isolation frequency. However, the molecular

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basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small

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intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential

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virulence factors were determined using two-dimensional electrophoresis （2-DE）coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential

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expression (fold change>1.2), we identified 31 different proteins, of which the expression

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abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained

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DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in

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virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels. Key words: Cronobacter sakazakii, Comparative proteomic analysis, Histologic analysis, Virulence factors

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ACCEPTED MANUSCRIPT Introduction Cronobacter species are important foodborne pathogens associated with invasive infections

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(septicemia, meningitis, and necrotizing enterocolitis) through the consumption of contaminated

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powdered infant formula (Biering et al., 1989; Gurtler et al., 2005; Nazarowec-White and Farber, 1999; van Acker et al., 2001). The virulence differences of Cronobacter isolates were firstly studied by Pagotto et al (2003). Furthermore, Townsend et al. (2007) demonstrated that

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Cronobacter could invade rat capillary endothelial brain cells (rBCEC4) in vitro and the

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persistence of Cronobacter in macrophages varied between Cronobacter strains. Cronobacter from an outbreak infection in France attached and invaded Caco-2 human epithelial cells and rat

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brain capillary endothelial cells (Townsend et al., 2008). The outer membrane proteins such as

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ompA and ompX were required for adhesion or invasion of Cronobacter into Caco-2 and INT-407

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(Kim and Loessner, 2008; Mittal et al., 2009; Mohan Nair and Venkitanarayanan, 2007; Singamsettyet et al., 2008). In addition, zpx gene encoding the cell-bound zinc-containing

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metalloprotease might be important in dissemination of the pathogen into the systemic circulation (Kothary et al., 2007). To date, Cronobacter consists of seven species of C. sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter condiment, Cronobacter universalis (Iversen et al., 2008; Joseph et al., 2012). Kucerova et al. (2010) reported that only strains from C. sakazakii, C. malonaticus, and C. turicensis were associated with infantile infections, and C. sakazakii is the most common species in terms of isolation frequency (Muller et al., 2013). C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis using PubMLST database, but no particular virulence traits have been 3

ACCEPTED MANUSCRIPT determined in C. sakazakii CC4 compared to other sequence types (Forsythe et al., 2014). As the part of LPS of Gram-negative bacteria, structures of O antigens in C. sakazakii have been

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identified (Arbatsky et al., 2010, 2012; Maclean et al., 2009, 2010). Although several genes such

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as zpx, ompA, and ompX, have been identified to be implicated in invasion or adherence of Cronobacter, little focus has been placed on the molecular basis of virulence differences among C. sakazakii isolates.

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The global techniques such as comparative proteomic analysis have been widely applied in

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elucidation of molecular basis involved in virulence differences (Cuervo et al., 2008; Donaldson et al., 2011; Mattow et al., 2003). Regulatory factors related to virulence might happen

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post-translationally, so measurement of mRNA levels might give incomplete information. In

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contrast, the analysis of global changes by a comparative proteomic assay seems reasonable for

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revealing differentially expressed proteins between virulent and attenuated isolates. In this study, on the basis of pathological and histologic analyses of virulent isolate (G362)

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and attenuated isolate (L3101), we used 2-DE technology coupled with MAIDI/TOF/TOF mass spectrometry for comprehensive proteomic analysis and identification of differential expression proteins between G362 and L3101. As a result, we found proteins that might play important roles in virulence differences among C. sakazakii isolates, and such information might contribute to revealing molecular basis of its pathogencity.

Materials and methods C. sakazakii isolates used in this study In table 1, virulence differences among 31 C. sakazakii isolates from food samples obtained by the ISO method combined with PCR assay targeting rpoB (Stoop et al., 2009) were determined 4

ACCEPTED MANUSCRIPT through intraperitoneal injection (1.2×106 cfu/mouse) into one week-old mice from the animal experimental center of Anhui Provincial Hospital.

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Analysis of slices of tissues (small intestine, kidney, and liver)

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C. sakazakii isolate (100 µl) G362 and L3101 (with 3.5×108 cfu/ml) was injected into one week-old mice via oral route of infection for every two days. After fifteen days, the mice were killed after ether treatment for collection of small intestine, kidney, and liver samples. Then, the

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tissues were fixed in 10% buffered formalin (Sangon, Shanghai), then routinely dehydrated by

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ethanol (Sangon, Shanghai) with different concentrations (v/v), and thereafter embedded by paraffin (Sangon, Shanghai). Tissues sections of 4-5 µm were cut and stained with hematoxylin

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and eosin (HE, Sangon, Shanghai). Histological analysis was performed by one pathologist who

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was blind to the treatment. The corresponding samples from the mouse treated with normal saline

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were analyzed as negative controls.

Preparation of whole cell proteins of C. sakazakii isolates

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Two C. sakazakii isolates were inoculated into Luria-Bertani (LB, Haibo, Qingdao) at 37 oC for 16 h. The whole cell proteins were extracted from 1.0 g wet cells using protein extraction kit 786-258 according to the manufacturer’s instructions (G-Biosciences, St Louis, MO. USA) and the extracted proteins were treated using Perfect-FOCUS™ (G-Biosciences, St Louis, MO. USA). The concentration of proteins was determined using non-interference protein concentration determination kit SK3071 (Sangon, Shanghai).

2-DE conditions For IEF, the protein samples (800 µg) were mixed with rehydration solution (with IPG buffer pH 3–10). The ImmobilineTM DryStrip IPG strips (13 cm, pH 3–10) were rehydrated at 30 V for 5

ACCEPTED MANUSCRIPT 12 h and continuously focused for 1 h at 500 V, 1 h at 1000 V, 8 h at 8000 V, 4h at 500 V at 20 oC under mineral oil. Then, the IPG strips were incubated for 20 min in 2D Equilibration Buffer

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（SD6030 with 1% DTT, Sangon, Shanghai) followed by 2D Equilibration Buffer（SD6030 with

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2.5% IAA, sangon, Shanghai）for 20 min. After the equilibration steps, the strips were transferred to 13 cm ×13 cm 12.5 % SDS-PAGE gels for the second dimension. Electrophoresis was performed at 30 A at 10 oC for 30 min. Proteins spots were stained with low background Silver

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Stain Kit (Sangon, Shanghai) according to the manufacturer’s instruction.

Image analysis and statistics

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The gels for triplicates were scanned with a densitometric Image Scanner (GE Healthcare) and

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the raw images were analyzed using the Image MasterTM platinum version 7.0 (GE Healthcare).

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For each comparison (G362 and L3101), the 2-D gels in triplicate were analyzed. To sufficiently screen for virulence factors, proteins with intensities of >1.2 fold and p values <0.05 are

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considered significant differences between G362 and L3101 and were identified with MAIDI/TOF/TOF mass spectrometry.

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Identification of proteins by MS To identify differentially expressed proteins, the respective spots were excised from the 2-DE gel and subjected to in-gel digestion as described by Lehner and Riedel (Riedel and Lehner, 2007). The excised proteins were analyzed on a 4700 proteomics analyzer MALDI-TOF/TOF (Applied Biosystem) as described by Riedel and Lehner (Riedel and Lehner, 2007). Protein identities were based on a combination of peptide fingerprint and MS/MS spectra. MS and MS/MS data were searched using MASCOT version 1.9.05 (Matrix Science) as search engine against the non-redundant NCBI database. Global Proteomics Server (GPS) explorer software (Applied Biosystem) was used for submitting data acquired with MALDI-TOF/TOF mass 6

ACCEPTED MANUSCRIPT spectrometer for database searching.

Quantitative analysis of partial genes using RT-PCR

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For real-time PCR, primers for ompA, Dps and LuxS genes are listed in Table 3 and 16S

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rRNA gene was used as internal control. The RNA was extracted from 1.2 ml overnight culture of C. sakazakii isolates using bacterial RNA extraction kit (BIOMIGA, USA). Then, cDNA was obtained using first-strand cDNA synthesis kit (BIOMIGA, USA) according to the manufacturer

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instructions. All samples were amplified in reactions containing 2 µl cDNA (25ng/µl), 10 µl 2×

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SYBR Green qPCR mix (Giagen, Beijing), 0.2 µM for each primer, and RNase-free water in a final volume of 20 µl. The PCR program was initiated at 95 oC for 1 min, followed by 40 cycles at

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95 oC for 10 s, 55 oC for 20 s, 72 oC for 25 s. The relative expression of targeted genes between

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G362 and L3101 was calculated using △△Ct method.

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Results and discussion

C. sakazakii is the most common species involved in infant infections (Kucerova et al., 2010).

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In this study, based on the intraperitoneal injection with 31 C. sakazakii isolates, the virulence differences between isolate G362 and L3101 were preliminary determined. In table 1, five mice died with 64h for each isolate infection. Two isolates (G362 and G581) caused death of mice within 14h, while all of five mice died with L3101 infection until 64h. Then G362 and L3101 were selected for further study and the histopathological analysis was used to confirm the observation in the experiment of intraperitoneal injection. From Fig.1, the flattening and vacuolation of the mucosal layer with G362 and L3101 infections were clearly observed, which was consistent with findings described by Townsend et al (2008). In addition, the flattening and vacuolation of the mucosal layer in G362 infection was more prominent than those with L3101 7

ACCEPTED MANUSCRIPT infection. Likely, liver tissues with G362 and L3101 infections showed marked fatty degeneration of liver cells and the spotty necrosis of liver cells was only observed in sample with G362

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infection. Additionally, glomerular burst and tubulorrhexis in kidney tissue with G362 were more

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prominent than those with L3101 infection. The significant morphological change in small intestine, liver, and kidney further reveals the virulence differences between G362 and L3101. Thereafter, whole cell proteins from G362 and L3101 were subjected to 2-DE analysis and

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the proteomic profiles were shown in Fig 2. To sufficiently screen for virulence factors, proteins

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with intensities of >1.2 fold were identified with MAIDI/TOF/TOF mass spectrometry. The proteomic analysis was showed in Table 2. An increased expression of proteins such as OmpA,

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Dps, LuxS as shown in Fig.3 was observed in strain G362, compare to that of L3101. In Fig.4, the

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expression abundance of ompA, Dps, and LuxS genes in G362 was 1.63, 3.49, and 4.85 fold

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changes of those in L3101 using quantitative PCR assay respectively, which was consistent with the results by 2D technology.

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OmpA in Cronobacter is required in adhesion or invasion to Caco-2 and INT-407 cells (Ingamsettyet et al., 2008; Mittal et al., 2009; Mohan Nair and Venkitanarayanan, 2007). Additionally, OmpX also played a critical role in basolateral invasion of C. sakazakii ATCC 29544 (Kim et al., 2010). The findings presented here further confirmed the functions of outer membrane protein A in virulence of C. sakazakii. Expression of DNA starvation/stationary phase protection (Dps) protein in G362 was approximately 9.4 fold greater than those seen for L3101. The Dps of C. sakazakii is 89%, 91%, and 92% similar at the amino acid level to that of E. coli, Salmonella enterica, and Klebsilella pneumoniae from NCBI alignment. Dps has been shown to enhance oxidative stress resistance and virulence (Halsey et al., 2004) in Salmonella and has been shown to 8

ACCEPTED MANUSCRIPT protect stationary grown E. coli cells from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock (Nair and Finkel, 2004). Dps is also considered to be

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involved in the virulence of Campylobacter jejuni (Heoret et al., 2011). Dps in Ralstonia

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solanacearum contributes quantitatively to host plant colonization and bacterial wilt disease (Colburn-Clifford et al., 2010). Interestingly, S-ribosylhomocysteinase (encoded by LuxS gene) was up-regulated in isolate G362. LuxS/AI-2 quorum sensing was reported to play important roles

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in bacterial virulence. From NCBI data, the LuxS identities of 90%, 91%, 92%, and 98-99% with

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E. coli, Salmonella enteric, Klebsilella pneumaniae and other Cronobacter species were observed. The luxS deletion mutant displayed attenuated production of virulence Stx2e in E. coli (Yang et al.,

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2014). Production of shiga toxin by a luxS mutant of E. coli O157: H7 in vivo and in vitro was

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also described (Jeon and Itoh, 2007). Palaniyandi et al. reported that LuxS contributed to the

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invasion and lethality in Avian pathogenic Escherichia coli (APEC) O78: K80: H9 strain (Palaniyandi et al., 2013).

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In addition, there are other proteins whose increased expression was also observed in strain G362 compared to strain L3101. For example, an increased expression on LysM-containing proteins was also seen in strain G362 compared to strain L3101. Such proteins from prokaryotes and eukaryotes comprise truly secreted proteins and outer membrane proteins, and lipoproteins (Buist et al., 2008) and have been shown to be involved in enhanced virulence in human bacterial pathogens such as Staphylococcus aureus (Buist et al., 2008), Listeria monocytogenes (Bierne and Cossart, 2007), and Escherichia coli (Bateman and Bycroft, 2000). ATPase proteins play important roles in virulence of bacteria (Chastane et al., 2004; de Oliveira et al., 2011; Nair et al., 2000; Yuan et al., 2007). The clpB plays an important role in cellular invasion and/or virulence of 9

ACCEPTED MANUSCRIPT Porphyromonas gingivalis (Yuan et al., 2007) and L. monocytogenes (Chastane et al., 2004). In Enterococcus faecalis, ClpB is involved in its thermotolerance and virulence (de Oliveira et al.,

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2011) and ClpC in L. monocytogenes contributes to adhesion and invasion and regulates

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expression of virulence factors InlA, InlB, and ActA (Nair et al., 2000). In addition, a ClpC ATPase required for stress tolerance and in vivo survival of L. monocytogenes was determined (Rouquette et al., 1996). ATP-binding cassette (ABC) transporters are integral membrane proteins

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that carry a variety of substrates across biological membranes（Davidson et al., 2008）. The

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increased expression of glutamine ABC transporter (Maclean et al., 2009) and cystine ABC transporter proteins (Arbatsky et al., 2012; Cuervo et al., 2008; Mattow et al., 2003) was observed

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for strain G362 compared to that of L3101; and suggests that ABC transporters are essential in C.

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sakazakii cell viability, virulence, and pathogenicity (Lewis et al., 2012). Sugar binding to ABC

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proteins can trigger a signaling response that results in increased virulence gene expression (Kemner et al., 1997). In Neisseria gonorrhoeae, L-cystine limitation leads to reduction in

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virulence (Bulut et al., 2012).

Additionally, we also found that 3-oxoacyl-[acyl-carrier-protein] synthase and malate dehydrogenase proteins were expressed only in isolate L3101. To our knowledge, these two proteins were not found to be related with bacterial virulence. In summary, the potential virulence factors such as Dps, OmpA, and LuxS were successfully identified in this study. These proteins might be involved in virulence of C. sakazakii through enhancing adherence or invasion to targeting issues, resistance or tolerance to environmental stresses and host immunologic system. Although proteins possibly related with virulence were identified, the detailed regulation mechanism(s) for these proteins remains to be 10

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Acknowledgments

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We gratefully acknowledge the financial support of the National Natural Science Foundation of

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China (31201292) and Guangdong Province, Chinese Academy of comprehensive strategic cooperation project (2012B090400017). Thank Prof. Jianpeng Hu for technical help on histologic

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analysis.

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M-T., Pellegrini,

E., Bolla,

J-M., Tascon,

R.

I., Vazquez-Boland,

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Rouquette,

J-A., Berche, P., 1996. Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes. Molecular Microbiology 21, 977-987. Singamsetty, V. K., Wang, Y., Shimada, H., Prasadarao, N. V., 2008. Outer membrane protein A expression in Enterobacter sakazakii is required to induce microtubule condensation in human brain microvascular endothelial cells for invasion. Microbial Pathogenesis 45, 181-191. Stoop, B., Lehner, A., Iversen, C., Fanning, S., Stephan, R., 2009. Development and evaluation of rpoB based PCR systems to differentiate the six proposed species within the genus 16

ACCEPTED MANUSCRIPT Cronobacter. International Journal of Food Microbiology 136, 165–168. Theoret, J. R., Cooper, K. K., Glock, R. D., Joens, L. A., 2011. A Campylobacter jejuni Dps

IP

T

Homolog Has a Role in Intracellular Survival and in the Development of Campylobacterosis

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in Neonate Piglets. Foodborne Pathogens and Disease 8, 1263-1268.

Townsend, S., Hurrell, E., Forsythe, S., 2008. Virulence studies of Enterobacter sakazakii isolates associated with a neonatal intensive care unit outbreak. BMC Microbiology 8, 64.

NU

Townsend, S. M., Hurrell, E., Gonzalez-Gomez, I., Lowe, J., Frye, J. G., Forsythe, S., Badger, J.

MA

L., 2007. Enterobacter sakazakii invades brain capillary endothelial cells, persists in human macrophages influencing cytokine secretion and induces severe brain pathology in the

D

neonatal rat. Microbiology 153, 3538–3547.

TE

van Acker, J., De Smet, F., Muyldermans, G., Bougates, A., Naessens, A., Lauwers, S., 2001.

CE P

Outbreaks of necrotizing enterocolitis associated with Entrobacter sakazakii in powdered milk formula. Journal of Clinical Microbiology 39, 293–297.

AC

Yang, Y., Zhou, M. M., Hou, H. Y., Zhu, J., Yao, F. H., Zhang, X. J., Zhu, X. F., Hardwidge, P. R., Zhu, G. Q., 2014. Quorum-sensing gene luxS regulates flagella expression and Shiga-like toxin production in F18ab Escherichia coli. Canadian Journal of Microbiology 60, 355–361. Yuan, L. H., Rodrigues, P. H., Belanger, M., Jr, W. D., Progulske-Fox, A., 2007. The Porphyromonas gingivalis clpB gene is involved in cellular invasion in vitro and virulence in vivo. FEMS Immunology and Medical Microbiology 51, 388-398.

17

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

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IP

T

Fig. 1

18

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 2

19

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

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IP

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Fig.3

20

ACCEPTED MANUSCRIPT Fig.4 L3101 G362

T IP

5

SC R

4 3

NU

2 1

MA

Relative expression of genes

6

0 LuxS

ompA

AC

CE P

TE

D

Gene Symbols

21

Dps

ACCEPTED MANUSCRIPT Fig.1. Histological analysis of tissues with 100µl （3.5×108 cfu/ml）C. sakazakii infections by gavage in mice using HE staining.

IP

T

Small intestine: A and B indicate the vacuolation and flattening of the mucosal layer respectively;

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Liver: A and B indicate fatty degeneration and spotted necrosis of liver;

Kidney: A and B indicate glomerular burst and tubulorrhexis respectively. Bar: 5µm.

NU

Fig.2. Whole cell proteomic profiles of virulent isolate (G362) and attenuated isolate (L3101) by

MA

two-dimensional gel electrophoresis (pH: 3-10).

D

Fig.3 Repeated experiments of partial differential expression proteins between G362 and L3101.

TE

OmpA: outer membrane protein A, Dps: DNA protection during starvation protein, LuxS:

CE P

S-ribosylhomocysteinelyase.

Fig.4 Relative fluorescence quantitative PCR of partial genes between G362 and L3101

AC

OmpA: outer membrane protein A, Dps: DNA protection during starvation protein, LuxS: S-ribosylhomocysteinelyase.

22

ACCEPTED MANUSCRIPT Table.1 The animal toxicity test in vivo of 31 C. sakazakii isolates1 C. sakazakii isolates

Time of death after injected infection(h) 10

14

18

22

26

30

34

38

42

46

50

54

60

64

2

1

1

0

0

0

1

0

0

0

0

0

0

0

0

G 121

1

1

0

2

0

1

0

0

0

0

0

0

0

0

0

G132

0

0

4

0

1

0

0

0

0

0

G371

2

0

1

1

0

0

0

1

0

0

G581

1

2

2

0

0

0

0

0

0

G441

0

0

1

1

0

0

1

1

0

2

0

0

0

0

1

2

0

2

0

0

L381

1

1

1

0

1

0

1

L481

0

0

0

3

0

0

1

L3101

0

0

0

0

0

0

G051

0

0

2

0

0

G142

1

3

0

1

0

G271

0

0

1

0

G351

2

1

1

0

G282

1

1

2

1

G312

1

1

1

G151

0

1

0

L982

2

1

1

L114

0

0

G481

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

IP

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

1

0

0

0

0

1

1

0

2

0

1

0

1

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

3

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

0

0

0

0

0

0

0

0

2

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

2

2

0

0

0

0

0

0

0

0

1

0

2

1

0

0

1

0

0

0

0

0

0

0

0

2

0

2

0

0

0

1

0

0

0

0

0

0

0

0

0

2

1

1

0

0

0

1

0

0

0

0

0

0

0

0

2

1

1

0

0

0

0

0

0

0

0

0

0

0

0

1

2

2

0

0

0

0

0

0

0

0

0

0

0

1

0

2

2

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

0

0

0

0

1

0

0

0

0

1

0

0

1

0

0

0

1

0

0

0

1

0

0

0

1

1

G084

0

1

2

1

0

0

0

0

1

0

0

0

0

0

0

L5101

0

0

0

2

0

0

0

1

0

0

1

0

0

0

1

L173

1

3

0

0

0

0

1

0

0

0

0

0

0

0

0

L781 G071 L681 G032 L541 L492

AC

G381

MA

NU

1

0

0

D

2

L073

CE P

G362

0

TE

﹟

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L 2101

T

6 ﹟

1: The experiments in vivo were carried out by intraperitoneal injection. C. sakazakii isolates were inoculated into LB and incubated at 37 oC for 14 h. Then, bacterial liquid with different concentrations was made by ten-fold and each mouse was injected into 1.2×106 cfu/mouse by intraperitoneal injection. We observed the death of mice and take record. The speed to death was considered as the index to judge the virulence of isolates. The biggest speed means the strongest virulence. ﹟

:G: isolates from dry fungus samples; L: isolates from frozen food samples

23

ACCEPTED MANUSCRIPT Table.2. Identification and characterization of differential expression proteins by MALDI/TOF/TOF MS between G362 and L3101

30

107

Malate dehydrogenase

27

228

DNA protection during starvation protein

25

342

Sequ ence

pIc

(Da)

cover age

d

5.91

locati on

Express ion of G362/L 3101 (fold change)

41%

cytoplas mic

↑(1.29)

5.42

325 08

12%

Cytopla smic

Only in isolate L3101

156934693

5.49

185 78

25%

Cytopla smic

↑(1.27)

156935752

TE

CE P

Protei ns

158 18

156933549

D

Putative LysM domain/BON superfamily protein

Mas s

T

scor eb

No.

Calcul ated

IP

N o.a

GI

SC R

Mas cot

NU

Sp ot

MA

Identified protein

31

294

468157

6.61

336 78

11%

Cytopla smic

↑(1.53)

Phosphoenolpyruv ate carboxykinase

24

154

156936435

5.04

598 88

5%

Cytopla smic

↑(1.29)

ATPase with chaperone activity

29

899

152973457

5.66

102 522

12%

Cytopla smic

↑(1.25)

Outer membrane protein A

28

705

156934557

5.03

384 34

31%

Outer membra ne

↑(1.30)

Hypothetical protein

34

196

156933457

8.57

205 92

15%

-

Only in isolate L3101

Putative outer membrane protein

33

368

494973783

8.57

203 78

22%

Outer membra

↓(11.4)

AC

Glyceraldehyde-3-p hosphate dehydrogenase A

24

ACCEPTED MANUSCRIPT ne 32

599

308827063

5.66

104 537

10%

Alkyl hydroperoxideredu ctase protein C

22

587

495056876

5.13

208 95

44%

Glutathionine S-transferase

18

519

495049798

Ribosomal RNA small subunit methyltransferase G

17

484

495054808

Glutamine ABC transporter

16

480

Arginine ABC transporter

15

MA

IP

SC R

Cytopla smic

↓(1.20)

35%

Cytopla smic

↓(1.30)

6.3

232 34

41%

Cytopla smic

↑(1.70)

9.06

284 84

24%

periplas mic

↑(1.50)

8.64

268 35

38%

periplas mic

↑(1.40)

TE

D

156934694

↑(1.40)

225 63

NU

5.93

Cytopla smic

T

Putative ATPase with chaperone activity, clpB

494981331

Cystine ABC transporter

20

938

156933484

8.96

290 29

33%

periplas mic

↑(4.50)

Cystine ABC transporter

21

940

156933484

8.96

290 29

33%

periplas mic

↓(3.40)

Osmoprotectant uptake system substrate-binding protein osmF binding protein YehZ

14

494

260598592

7.74

324 39

27%

periplas mic

↑(1.60)

Putative ATPase with chaperone activity, clpC

13

898

152973457

5.66

104 429

14%

Unkown

↑(1.20)

Putative ATPase with chaperone activity, clpC

10

119 3

152973457

5.66

104 429

14%

Unkown

↑(1.90)

AC

CE P

640

25

ACCEPTED MANUSCRIPT Hypothetical protein

1

9

- 345 86

516029546

4

2

0

Glyceraldehyde-3-p hosphate dehydrogenase A

11

358

152969751

6.61

356 13

Fructose-bisphosp hatealdolase

8

881

156932986

5.69

392 54

35%

-

% cytoplas mic

↑(1.80)

cytoplas mic

↓(1.30)

9

959

156932986

5.69

392 54

42%

cytoplas mic

↑(1.74)

Folate-dependent protein

25

396

495053100

5.34

362 25

22%

cytoplas mic

↓(1.58)

Transaldolase

7

780

156932986

5.71

356 01

35%

cytoplas mic

↑(1.24)

Carbamoyl-phosph ate synthetase glutamine chain

2

287

495057684

5.60

426 32

9%

cytoplas mic

↓(1.4)

3-oxoacyl-[acyl-car rier-protein] synthase

3

495052045

5.27

428 14

21%

cytoplas mic

Only in isolate L3101

Alcohol dehydrogenase

1

739

495052045

6.20

961 57

13%

cytoplas mic

↑(1.43)

S-ribosylhomocyste inelyase

23

104

156932785

5.42

192 24

8%

cytoplas mic

↑(1.60)

Putative YfdX

36

234

372488163

9.03

323 01

12%

cytoplas mic

↑(1.22)

Small HspC2 heat shock protein

35

297

495055484

9.22

263 21

25%

Unknow n

↑(1.66)

Cystine ABC transporter

19

484

495051223

8.48

278 31

25%

periplas mic

↑(1.45)

Phosphoserine aminotransferase

5

985

156934603

5.30

396 02

36%

cytoplas mic

↓(1.43)

D TE 700

CE P

AC

MA

Fructose-bisphosp hatealdolase

NU

SC R

IP

T

17%

↑ (1.90)

26

ACCEPTED MANUSCRIPT 4

403

156935403

5.68

627 29

14%

Unknow n

↓(1.28)

DNA protection during starvation protein

26

141

156934693

5.49

185 89

14%

Cytopla smic

↑(7.70)

IP

SC R

a) The number refers to the spot numbers as given in Fig.2.

T

Acetolactate synthase

b) MOWSE scores of the highest confident matches (P<0.05).

c) pI, the predicted isoelectric point calculated from the protein sequence.

AC

CE P

TE

D

MA

NU

d) The predicted molecular weight calculated from the protein sequence.

27

ACCEPTED MANUSCRIPT Table 3. Primers for real-time PCR in this study Genes

Primers(5’-3’)

Sizes(bp)

OmpA

F-ctgggccgcatgccgtataa R-cgccagcgaataccggagaa F-tatgcgcggcgctaacttcat R-acgatccgccagctctttcag F-tcgatatctccccgatgggctg R-cgcctacgccacgttcgataa F-acgagtggcggacgggtga R-tcagttccagtgtggctgg

205

T

238

AC

CE P

TE

D

MA

NU

16S rRNA

231

IP

LuxS

214

SC R

Dps

28

ACCEPTED MANUSCRIPT Highlights ►The virulence differences of C. sakazakii isolates were determined by animal

IP

T

experiment and histological analysis.

SC R

►Proteomic profiles of virulent and attenuated isolates were firstly developed by 2-DE.

MAIDI/TOF/TOF coupled with MS.

NU

►Potential virulence factors such as Dps, OmpA, LuxS were identified by

MA

► Expression abundance of Dps, OmpA, and LuxS genes in G362 were increased

AC

CE P

TE

D

using qPCR

29