The PhoP/PhoQ two-component signal transduction system is an important regulator of stress responses and virulence in most gram-negative bacteria, but the characterization of PhoP/PhoQ in Shigella has not been thoroughly investigated. In the present study, we found that deletion of phoPQ (ΔphoPQ) from Shigella flexneri Recombinants 2a 301 (Sf301) resulted in a significant decrease (reduced by more than 15-fold) in HeLa cell and Caco-2 cell invasion, and less inflammation. . (- or +) compared to Sf301 (+++) in the Sereny guinea pig test. In low Mg2+ medium (10 μM) or pH 5 medium, the ΔphoPQ strain exhibited growth deficiency compared to Sf301.

The ΔphoPQ strain was more sensitive than Sf301 to polymyxin B, an important antimicrobial agent for the treatment of multidrug-resistant gram-negative infections. By comparing the transcriptional profiles of ΔphoPQ and Sf301 using DNA microarrays, 117 differentially expressed genes (DEGs) were identified, which were involved in Mg2+ transport, lipopolysaccharide modification, acid resistance, bacterial virulence, respiratory and energy metabolism.

Based on the reported PhoP box motif [(T/G)GTTTA-5nt-(T/G)GTTTA], we examined 38 suspected PhoP target operons in S. flexneri, and 11 of them (phoPQ, mgt, sly, you, yrbL, icsA, yhiWX, rstA, hdeAB, page and shf–rfbU-Virk-msbB2) were shown to be PhoP-regulated genes according to electrophoretic mobility shift assays and β-galactosidase assays. One of these PhoP-regulated genes, icsA, is a well-known virulence factor in S. flexneri. In conclusion, our data suggest that the PhoP/PhoQ system modulates the virulence of S. flexneri (in an ice-dependent manner) and the stress responses of Mg2+, pH, and antibacterial peptides.

Statement of Ethics

All guinea pig infection procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University College of Basic Medical Sciences (IACUC Animal Project Number 20140226-022-Qu) in accordance. with the national guidelines (Regulations for the Administration of Experimental Animal Related Affairs, China).

Bacterial strains, plasmids and growth conditions

S. flexneri 2a 301 (Sf301, GenBank accession number AE005674) was kindly provided by Pr. Qi Jin (MOH Key Laboratory of Pathogen Systems Biology, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Faculty of Medicine). Peking Union Medicine, Beijing, China). Bacterial strains and plasmids were used in this study. S. flexneri and E. coli were grown in Luria-Bertani medium (LB; Oxoid, Basingstoke, UK) at 37°C. Antibiotics were used at the following concentrations: ampicillin (100 μg/mL), kanamycin (50 μg/mL), tetracycline (10 μg/mL), and gentamicin (50 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA).

Test and pathological examination of S. flexneri Sereny

The virulence of S. flexneri was determined by the Sereny guinea pig keratoconjunctivitis test (Sereny, 1957). Female guinea pigs (6 weeks old, about 300 g) were inoculated with 109 Colony Forming Units (CFU) of bacteria per eye (six guinea pig eyes in each group) and observed at 24, 48 and 72 h. LB inoculation served as a negative control. Bacterial-induced keratoconjunctivitis was scored as follows: −, no disease or mild irritation; +, mild conjunctivitis or late development and/or rapid disappearance of symptoms; ++, keratoconjunctivitis without purulence; and +++, fully developed keratoconjunctivitis with purulence.

At 72 h post-inoculation, guinea pigs were euthanized with pentobarbital (40 mg/kg) and the eyes were removed and fixed in 4% formalin in PBS (pH 7.2). After hematoxylin and eosin (H&E) staining, the eye sections were examined under a microscope.

Bacterial growth curves under low Mg2+ conditions, acidic pH and presence of polymyxin B

The growth curves of the strains were determined by measuring the OD600 with an Eppendorf spectrophotometer at 60 min intervals over a period of 14 h. For the low Mg2+ growth assay, N medium containing 0.1 M Tris-HCl (pH 7.4), 38 mM glycerol, 0.1% (w/v) casamino acids, 0.37 KCl was used. g/l, K2SO4 0.087 g/l, 0.99 g/l (NH4)2SO4 and 0.14 g/l KH2PO4 (Barchiesi et al., 2012).

Overnight cultures of bacterial strains were inoculated into N medium supplemented with 10 μM or 10 mM MgCl2 (at a dilution of 1:50) and incubated at 37 °C with shaking. To evaluate the acid resistance of the bacteria, glucose E broth containing 0.2 g/l MgSO4•7H2O, 2 g/l citric acid, 13.1 g/l K2HPO4•3H2O, 3 0.5 g/l Na(NH4)HPO4•4H2O and 0.4% glucose. Overnight cultures were inoculated into glucose E broth at pH 7 or pH 5 (at a 1:50 dilution) and incubated at 37°C with shaking (Barchiesi et al., 2012).

For the polymyxin B resistance assay, overnight cultures were inoculated in LB, shaken grown until the OD600 reached 0.6, then the bacteria were diluted in sterile 0.85% saline to approximately 5%. × 103 cells per ml and exposed to different concentrations of polymyxin B (5, 10, 20 and 40 μg/ml) for 1 h at 37 °C. Surviving bacteria were determined by plating on LB agar plates in triplicate. Survival rate was calculated as the number of bacteria treated with polymyxin B divided by that of the untreated control. All experiments were repeated at least three times.

DNase I fingerprint assay

DNase I fingerprint assays were performed according to Wang et al. (2012). The promoter regions of your, mgt, and shf were amplified with the primers listed in Table 2 and cloned separately into pUC18BT vector (Shanghai Biotechnology Corporation, China), which was then used as a template for the preparation of FAM-labeled probes fluorescent.

The probes were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega) and quantified with a NanoDrop 2000C (Thermo Fisher Scientific Waltham, MA, USA). For each assay, 500 ng probes were incubated with different amounts of PhoP in 40 μl of binding buffer at 30 °C for 30 min. Then 10 μl of DNase I (0.01 units) (Promega) and 100 nmol of CaCl2 were added, incubated at 25 °C for 1 min and the reactions were stopped by adding 140 μl of DNase I stop solution (acetate).

200 mM unbuffered sodium, 30 mM sodium acetate EDTA, and 0.15% SDS). DNA samples were phenol/chloroform extracted, dissolved in 30 μl MilliQ water and loaded for capillary electrophoresis. Data was collected using the GeneScan-500 LIZ dye size standard (Applied Biosystems, Foster City, CA, USA).

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