Anaerobic regulation of Shigella flexneri virulence: ArcA regulates Fur and iron acquisition genes

Abstract

Invasion and plaque formation in epithelial monolayers are routinely used to assess the virulence of Shigella flexneri, a causative agent of dysentery. A modified plaque assay was developed to identify factors contributing to the virulence of S. flexneri under the anaerobic conditions present in the colon. This assay demonstrated the importance of the ferrous iron transport system Feo, as well as the global transcription factors Fur, ArcA, and Fnr, for Shigella plaque formation in anoxic environments. Transcriptional analyses of S. flexneri iron transport genes indicated that anaerobic conditions activated feoABC while repressing genes encoding two other iron transport systems, the ABC transporter Sit and the Iuc/Iut aerobactin siderophore synthesis and transport system. The anaerobic transcription factors ArcA and Fnr activated expression of feoABC, while ArcA repressed iucABCD iutA. Transcription of fur, encoding the iron-responsive transcriptional repressor of bacterial iron acquisition, was also repressed anaerobically in an ArcA-dependent manner.

FIG. 1.

Plaque formation by S. flexneri under aerobic and anaerobic conditions. Henle cell monolayers were infected with 10⁴ wild-type S. flexneri (SA100), avirulent Crb⁻ mutant (SA101), feoB iucD mutant (SA192), feoB sitA mutant (SM191), iucD sitA mutant (SA167), or feoB iucD sitA mutant (SM193) cells. The plates were incubated for 2 days in medium containing gentamicin under either aerobic (top) or anaerobic (bottom) conditions and stained to visualize plaque formation.

FIG. 2.

Effect of O₂ on expression of gfp fused to iron transport gene promoters. Wild-type S. flexneri containing plasmids carrying iron acquisition gene promoters fused to gfp were grown to mid-log phase in the presence or absence of oxygen, and relative fluorescence was measured. For each promoter, the condition with the highest fluorescence value was set at 100%. Experiments were performed in triplicate, and error bars represent 1 standard deviation.

FIG. 3.

Effect of anaerobiosis on expression of the iuc promoter. (A) Cultures were grown anaerobically to mid-log phase, and relative gfp expression from the iuc promoter of pEG6 in SA101 (WT) and MBF200W (arcA) was determined. The highest relative fluorescence value was set at 100%. Experiments were performed in triplicate, and error bars represent 1 standard deviation. (B) The S. flexneri iucABCD iutA chromosomal region is depicted, showing the relative positions of the Fur box, Shine-Dalgarno sequence (SD), and putative ArcA box. The putative ArcA box sequence in iuc is shown below the map, with bases matching the ArcA box consensus sequence in black and bases not matching the consensus in gray. Lowercase letters indicate bases not conserved in the ArcA box weight matrix.

FIG. 4.

Anaerobic expression of native and altered feo promoters. (A) Cultures were grown anaerobically to mid-log phase, and relative gfp expression from the feo (pMBfeo) and feo_Alt (pMBfeo_Alt) promoter fusions in SA101 (WT), MBF100W (fnr), MBF200W (arcA), and MBF300W (arcA fnr) was determined. The highest relative fluorescence value was set at 100%. Experiments were performed in triplicate, and error bars represent 1 standard deviation. (B) The S. flexneri feoABC chromosomal region shows the relative positions of the Fur box, Shine-Dalgarno sequence (SD), and putative ArcA box and Fnr box. The sequences resembling the putative ArcA box in the native and altered feo promoters are indicated below, with bases matching those of the putative ArcA box consensus sequence in black, bases not matching the consensus in gray, and bases changed in the altered promoter underlined. Lowercase letters indicate bases not conserved in the ArcA box weight matrix.

FIG. 5.

Predicted ArcA regulatory motif. Sequences of ArcA-regulated promoters were entered into the SeSiMCMC interface (http://favorov.imb.ac.ru/SeSiMCMC/), and the algorithm reported a conserved weight matrix for ArcA sequence recognition. The sequence logo was obtained by entering the weighted matrix derived from a multiple sequence alignment into the interface at http://weblogo.berkeley.edu/.

FIG. 6.

ArcA-dependent repression of fur transcription under anaerobic conditions. (A) S. flexneri SA100 (WT) with pCC1 (vector) and MBF200 (arcA) with pCC1 (vector) or pMBarcAccQE (pArcA) were grown to mid-log phase anaerobically with 1 μM IPTG. The level of fur mRNA was determined by RT-PCR. Experiments were performed in triplicate, and error bars represent 1 standard deviation. (B) The relative gfp expression levels obtained from fur (pMBfur) and fur_Alt (pMBfur_Alt) promoters in SA101 (WT) and MBF200W (arcA) are shown with the highest relative fluorescence value set at 100%. Experiments were performed in triplicate, and error bars represent 1 standard deviation. (C) The S. flexneri fur chromosomal region shows the relative positions of promoter elements involved in fur regulation, including the putative ArcA boxes, OxyR box, Crp box, and Fur box. The two putative ArcA boxes are indicated by the arrows, and their sequences are shown below. The bases in the native and altered fur promoters matching those of the putative ArcA box consensus sequence are shown in black. Bases not matching the consensus are in gray, and bases changed in the altered promoter are underlined. Lowercase letters indicate bases not conserved in the ArcA box weight matrix.

FIG. 7.

ArcA binds feo, fur, and iuc promoters. S. flexneri protein extracts prepared from strain MBF200 (arcA) containing either pCC1 vector (ArcA⁻) or pMBarcAccQE (ArcA⁺) were incubated with the indicated radiolabeled probes and anti-ArcA antibody. Samples were electrophoresed on a 5% polyacrylamide gel. Positions of probes (*) and supershifted bands (brackets) are indicated.

FIG. 8.

Fur and the anaerobic regulators ArcA and Fnr are important for plaque formation by S. flexneri. Henle cell monolayers were infected with 10⁴ CFU of the indicated S. flexneri strain. The plates were incubated for 2 days in medium containing gentamicin under either aerobic (A) or anaerobic (B) conditions and stained to visualize plaque formation. (A) SA100 (WT) and mutants MBF100 (fnr), MBF200 (arcA), and MBF300 (arcA fnr). (B) SM100 (WT), SM1301 (fur), or SM100/pMBfurQE (WT/pfur) incubated with or without 50 μM IPTG to induce fur expression.