Research Article Ygae Regulates out Membrane Proteins in Salmonella Enterica Serovar Typhi Under Hyperosmotic Stress

Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 374276, 9 pages http://dx.doi.org/10.1155/2014/374276 Research Article YgaE Regulates Out Membrane Proteins in Salmonella enterica Serovar Typhi under Hyperosmotic Stress Min Wang,1 Ping Feng,1 Xun Chen,2 Haifang Zhang,3 Bin Ni,3 Xiaofang Xie,1 and Hong Du1 1 Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China 2 Clinical Laboratory Center, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China 3 Department of Biochemistry and Molecular Biology, School of Medical Technology, Jiangsu University, Zhenjiang 212013, China Correspondence should be addressed to Hong Du; hong [email protected] Received 31 August 2013; Accepted 30 October 2013; Published 23 January 2014 Academic Editors: R. Tofalo and J. Yoon Copyright © 2014 Min Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Salmonella enterica serovar Typhi (S. Typhi) is a human-specific pathogen that causes typhoid fever. In this study, we constructed ΔygaE mutant and a microarray was performed to investigate the role of ygaE in regulation of gene expression changes in response to hyperosmotic stress in S. Typhi. qRT-PCR was performed to validate the microarray results. Our data indicated that ygaE was the repressor of gab operon in S. Typhi as in Escherichia coli (E. coli), though the sequence of ygaE is totally different from gabC (formerly ygaE)inE. coli. OmpF, OmpC, and OmpA are the most abundant out membrane proteins in S.Typhi.Herewereport that YgaE is a repressor of both OmpF and OmpC at the early stage of hyperosmotic stress. Two-dimensional electrophoresis was applied to analyze proteomics of total proteins in wild-type strain and ΔygaE strain and we found that YgaE represses the expression of OmpA at the late stage of hyperosmotic stress. Altogether, our results implied that YgaE regulates out membrane proteins in a time-dependent manner under hyperosmotic stress in S. Typhi. 1. Introduction lipid and protein components. The outer membrane proteins (OMPs), called porins, are characterized by a -barrel struc- S. Typhi is a human-specific pathogen, which produces ture and form water-filled channels for the passage of a large typhoid fever. Once ingested through contaminated water or variety of hydrophilic molecules [7–9]. food, S. Typhi invades intestinal epithelial cells and can enter In E. coli, the two general porins OmpF and OmpC 5 the host bloodstream and disseminate to deep organs. Lack- are among the most abundant OMPs (about 10 copies per ing of standard water supply and sanitation, typhoid remains cell) and serve as general pathways for the influx of small a major health problem in developing world [1–3]. In contam- molecules (e.g., molecular weight under 600). They consist of inated water or food the external osmolarity is in the order of three 16-stranded -barrels, each of which forms a channel 50 mM NaCl; however, in the lumen of the host intestine S. that is restricted in the middle due to the inward folding of Typhi cells are exposed to a significant increase in osmolarity: a loop (loop L3) [10]. The expression of E. coli porins has 300 mM NaCl in the small intestine [4]. Considering the sig- been extensively studied. The OmpF-OmpC balance is highly nificant morbidity and mortality associated with this disease regulated by different genetic control systems. Changes in [5], it is important to understand the gene regulation mecha- osmolarity profoundly affect expression of OmpF and OmpC. nisms in S. Typhi in response to hyperosmotic environments. OmpC is preferentially expressed in high osmolarity, whereas Theoutermembrane(OM)ofGram-negativebacteria OmpF expression is favored in low osmolarity [11]. Other constitutes the first permeability barrier that protects the factors, including local anesthetics [12], pH [13], and nutrition cells against environmental stresses including chemical and limitation [14]alsoinfluenceompF and ompC transcription biological attacks [6]. Simultaneously, it allows the selective in an EnvZ/OmpR-dependent manner. Noteworthy, growth uptake of essential nutrients and the secretion of metabolic conditions where nutrient levels are high, such as in mammal waste products. The OM is a sophisticated organization of intestinal tracts, favor the expression of OmpC, which has a 2 The Scientific World Journal Table1:Strainsandplasmidsusedinthisstudy. Strains or plasmid Relevant characteristics Source Strains Salmonella enterica serovar Typhi Wild-type strain; z66+ Gifu University [22] GIFU10007 ΔygaE GIFU10007; z66+ This study ΔygaE (pBAD) ΔygaE harboring pBAD plasmid; This study ΔygaE harboring pBAD-ygaE ΔygaE (pBADygaE) This study recombinant plasmid; E. coli SY372pir E. coli host strain of suicide plasmid Gifu University [23] Plasmids pGMB151 Suicide plasmid; sacB; Ampr Gifu University [23] pBAD/gIII Expression vector; Ampr Invitrogen pBADygaE pBAD/gIII containing ygaE gene This study Table 2: Primers used in this study. Name Sequence ygaE-F1A(+BamH I) 5 -TTGGATCCGGTACTGTCCCCATTATGT ygaE-F1B(+Sal I) 5 -CCGTCGACATAGCCTTTTTGATTCACC ygaE-F2A(+Sal I) 5 -TAGTCGACGAGATGCTGGAAGATAAAC ygaE-F2B(+BamH I) 5 -CTGGATCCTCTGCTACACTTTTCTTTG ygaE-PA(+Nco I) 5 -AACCATGGAGATGACCGCCCTTTCCCAA ygaE-PB(+Sal I) 5 -AAGTCGACCTACATTTTTCCTGCCAT P-ompF-A 5 -GGA ATACCGTACTAA AGCA P-ompF-B 5 -GATACTGGATACCGA AAGA P-ompC-A 5 -ATCAGA ACAACACCGCTAA P-ompC-A 5 -GTTGCTGATGTCCTTACC smaller channel than OmpF, thus limiting the influx of large stress and OmpA at the late stage of hyperosmotic stress in andchargedmoleculessuchasbilesaltsandantibiotics.Con- S. Typhi. versely, OmpF will be the major porin under ex vivo growth conditions with nutritional deficiency, as its larger pore will 2. Materials and Methods allow efficient influx of nutrients [15]. In contrast, expression of OmpC in S. Typhi is not influenced by osmolarity, while 2.1. Bacterial Strains and Conditions of Culture. The bacterial OmpF is regulated as in E. coli [16]. strains and plasmids used in this study are listed in Table 1. ∘ OmpA is another abundant OMP. It is monomeric, and it Bacteria were grown in LB broth at 37 Cwithshaking is unusual in that it can exist in two different conformations (250 rpm). As for antibiotic sensitivity assay, Muller Hinton [17]. A minor form of the protein, with an unknown number agar was used. For low environmental osmolarity, the growth of transmembrane strands, can function as a porin, but the medium contained a final concentration of 50 mM NaCl. For major, nonporin form has only eight transmembrane strands, hyperosmotic environment, Nacl was added into the medium and the periplasmic domain of this form performs a largely at a final concentration of 300 mM. The complemented structural role [18]. The function of OmpA is thought to strains were induced by L-arabinose (0.2% wt/vol). When contribute to the structural integrity of the outer membrane appropriate, ampicillin was added to the medium at a final along with murein lipoprotein [19] and peptidoglycan associ- concentration of 100 g/mL. ated lipoprotein [20]. In E. coli, gabC (formerly ygaE) was reported to belong to 2.2. Construction of the ygaE Deletion Mutant Strain. The the gabDTPC operon, and gabC is the repressor of the operon ygaE deletion mutant (ΔygaE)waspreparedbyhomologous [21]. The function of gab operon is mainly revolved in - recombination according to a previously described method aminobutyrate (GABA) catabolism but does not contribute to [24]withaygaE-deletion suicide plasmid lacking 327 bp of thecatabolismofanyothernitrogensource[21]. However, the the ygaE gene. The specific primers used for deletion of ygaE function of ygaE in S. Typhi has not been extensively studied. were listed in Table 2. The mutant strain was selected by In this work, we showed that ygaE regulates out membrane PCR as described previously [23], verified by sequencing, and proteins OmpF/OmpC at the early stage of hyperosmotic designated ΔygaE. The Scientific World Journal 3 2.3. Complement of ygaE in the ΔygaE Mutant Strain. Specific from three independent experiments. Student’s -test was primers ygaE-PA and ygaE-PB (Table 2) were designed to used for the statistical analysis. Differences were considered amplify a ygaE promoterless DNA fragment with pfu DNA statistically significant when was <0.05 in all cases. polymerase (Takara). Nco I and Sal I sites were added to the 5 -termini of primers ygaE-PA and ygaE-PB, respectively. 2.7. Measurement of Bacterial Growth. Wild-type and ygaE The amplicon was digested by Nco I and Sal I and inserted mutant were overnight cultured; then 200 L aliquots of into the expression vector pBAD/gIII (Invitrogen), which the culture were diluted to 20 mL fresh LB medium (with ∘ was predigested with the same restriction enzymes, to form NaCl concentration of 300 mM) and incubated at 37 Cwith the recombinant plasmid pBADygaE.Thepositiveplasmid shaking (250 rpm). The growth was measured every two Δ was verified by sequence analysis. ygaE was transformed hours using a BioPhotometer (Eppendorf). The measurement Δ with pBADygaE and designated as ygaE (pBADygaE). As was performed three times. Student’s -test was used for the Δ control, ygaE was transformed with pBAD and designated statistical analysis. Differences were considered statistically Δ Δ as ygaE (pBAD). Expression of ygaE

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