Dissecting virulence: systematic and functional analyses of a pathogenicity island

Abstract

Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach. These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease.

Fig. 1.

Both Ler and Orf11 are required for expression of LEE genes in CR. (A) Genetic organization of CR LEE (7). (B) Expression of Tir and EspB in WT CR and its 41 LEE mutants. Whole-cell lysates of bacteria grown in DMEM were analyzed by 10% SDS/PAGE and Western blot with anti-Tir and anti-EspB sera.

Fig. 2.

Orf11 and Orf10 regulate ler expression in CR. (A) Western blot with anti-Tir and anti-EspB sera of total lysates of bacteria grown in DMEM. Lane 1, WT CR; lane 2, Δorf11; lane 3, ΔlerΔorf11. Also shown are CR Δorf11 complemented by orf11 from CR (pCRorf11, lane 4), EHEC (pEHorf11, lane 5), or EPEC (pEPorf11, lane 6); and CR ΔlerΔorf11 double mutant complemented by CR ler (lane 7) or orf11 (lane 8). (B) Orf11 positively regulates ler expression. The transcriptional activity directed by the ler-cat fusion in pLEE1/Ler-CAT was determined in CR WT, Δler, Δorf10, and Δorf11 grown in DMEM for 6 h. The data are the average of three experiments. (C) Orf11 positively regulates the expression of LEE2 and LEE5 operons by activating ler expression. The activity directed by LEE2 (pLEE2-CAT) and LEE5 (pLEE5-CAT) transcriptional fusions was measured in CR WT, Δler, Δorf10, and Δorf11 as described above. (D) Orf10 acts as a negative regulator of LEE gene expression when expressed from a plasmid. Whole-cell lysates of WT CR carrying pCR2.1-TOPO (the cloning vector, lane 1), pCRorf10-2HA (2HA-tagged orf10, lane 2), pCRorf10 (CR orf10 with its own promoter, lane 3), pCRorf10_Plac (Plac-driven CR orf10, lane 4), and pCRorf10orf11 (CR orf10 and orf11 with their own promoter, lane 5) were analyzed as for A.

Fig. 3.

Type III secretion by WT CR and its 41 LEE mutants. (A) General protein secretion profile of CR and its mutants. (B) Tir and EspB secretion analyzed by Western blot with anti-Tir and anti-EspB sera. (C) Secretion profile of ΔsepL, Δrorf6, ΔescN (TTS mutant), and their double mutants. Secreted proteins were concentrated from supernatants of bacterial cultures grown in DMEM and analyzed by 12% SDS/PAGE and Coomassie blue G250 staining (A and C) or Western blot (B).

Fig. 4.

Identification of both LEE- and non-LEE-encoded proteins secreted by the LEE-encoded TTSS. (A) Effect of overexpressing CR orf11 on TTS in WT CR and its ΔsepL or Δrorf6 mutants. Secreted proteins were analyzed by 15% SDS/PAGE and Coomassie blue staining. The additional type III secreted proteins by ΔsepL and Δrorf6 carrying pCRorf11 are indicated by arrows and were characterized by proteomic analyses (Table 2 and Fig. 8). (B) A diagram showing locations of the O-islands encoding the six identified non-LEE effectors in the EHEC O157:H7 genome (3). Also shown are the locations of the Shiga toxin genes (stx), the LEE, the inv-spa-like TTSS, and the associated prophages (CP- and BP-933).