Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein

Abstract

Yersinia pestis, the causative agent of plague, must survive in blood in order to cause disease and to be transmitted from host to host by fleas. Members of the Ail/Lom family of outer membrane proteins provide protection from complement-dependent killing for a number of pathogenic bacteria. The Y. pestis KIM genome is predicted to encode four Ail/Lom family proteins. Y. pestis mutants specifically deficient in expression of each of these proteins were constructed using lambda Red-mediated recombination. The Ail outer membrane protein was essential for Y. pestis to resist complement-mediated killing at 26 and 37 degrees C. Ail was expressed at high levels at both 26 and 37 degrees C, but not at 6 degrees C. Expression of Ail in Escherichia coli provided protection from the bactericidal activity of complement. High-level expression of the three other Y. pestis Ail/Lom family proteins (the y1682, y2034, and y2446 proteins) provided no protection against complement-mediated bacterial killing. A Y. pestis ail deletion mutant was rapidly killed by sera obtained from all mammals tested except mouse serum. The role of Ail in infection of mice, Caenorhabditis elegans, and fleas was investigated.

FIG. 1.

Amino acid sequence alignment of Y. pestis KIM Ail with other Yersinia Ail/Lom family proteins. The amino acid sequences of Y. pestis KIM Ail (Yp Ail) and Y. enterocolitica Ail (Ye Ail) and the predicted amino acid sequences of the Y. pestis KIM Ail/Lom family y1682, y2034, and y2446 proteins are aligned. Amino acid residues in the four predicted surface-exposed loops are enclosed in boxes (loops 1 to 4). Identical residues are indicated by asterisks, strongly similar residues are indicated by colons, and weakly similar residues are indicated by periods. The alignment was generated using the CLUSTALW multiple-sequence alignment program (39).

FIG. 2.

Y. pestis Ail is highly expressed and required for survival in NHS. (A) Survival of wild-type and mutant strains of Y. pestis in 80% NHS. Bacteria grown overnight at 26 or 37°C (∼5 × 10⁶ bacteria) were incubated with 80% NHS or HIS for 1 h at 37°C. Aliquots were diluted and plated on TBA plates at 30°C. The percent survival was calculated as follows: average number of bacteria that survived exposure to NHS at 1 h/number of bacteria that survived exposure to HIS at 1 h × 100. (B) Silver-stained SDS-PAGE gel of outer membrane proteins isolated from the parent and mutant strains of Y. pestis. The location of the Ail (y1324) protein is indicated by an arrow. The positions of molecular mass standards (in kilodaltons) are indicated on the left. The strains used were Y. pestis strains KIM8-E (parent), KIM8-E Δy2446, KIM8-E Δy2034, KIM8-E Δy1324 (Δail), KIM8-E Δy1324 (Δail)/pAil, KIM8-E Δy1682, KIM8-E Δ4, and KIM8-E Δ4/pAil. mw std, molecular mass standards.

FIG. 3.

Ail is not expressed by Y. pestis strains grown at 6°C. (A) Survival of wild-type and mutant strains of Y. pestis in 80% NHS. Bacteria grown overnight at 6 or 26°C (∼5 × 10⁶ bacteria) were incubated with 80% NHS or HIS for 1 h at 37°C. Aliquots were diluted and plated on TBA plates at 30°C. The percent survival was calculated as follows: average number of bacteria that survived exposure to NHS at 1 h/number of bacteria that survived exposure to HIS at 1 h × 100. (B) Coomassie blue R-250-stained SDS-PAGE gel of whole bacterial cell proteins isolated from wild-type and mutant strains of Y. pestis. The location of the Ail protein is indicated by an arrow. The positions of molecular mass standards (in kilodaltons) are indicated on the left. The strains used were Y. pestis strains KIM8-E (parent), KIM8-E Δy1324 (Δail), and KIM8-E Δy1324 (Δail)/pAil. mw std, molecular mass standards.

FIG. 4.

Expression of Ail in E. coli provides protection from complement-dependent killing. (A) Survival of E. coli with or without plasmid pAil in 80% NHS. Bacteria grown overnight at 37°C (∼5 × 10⁶ bacteria) were incubated with 80% NHS or HIS for 1 h at 37°C. Aliquots were diluted and plated on LB plates at 37°C. The percent survival was calculated as follows: average number of bacteria that survived exposure to NHS at 1 h/number of bacteria that survived exposure to HIS at 1 h × 100. (B) Coomassie blue R-250-stained SDS-PAGE gel of whole bacterial cell proteins isolated from E. coli with or without plasmid pAil. The location of the Ail protein is indicated by an arrow. The positions of molecular mass standards (in kilodaltons) are indicated on the left. The strains used were E. coli strains DH5α, DH5α/pBluescript KS(−) (pKS⁻) (vector control), and DH5α/pAil.

FIG. 5.

Expression of Ail, but not expression of other Y. pestis Ail/Lom family proteins, provides protection against complement-dependent killing. (A) Coomassie blue R-250-stained SDS-PAGE gel of whole bacterial cell proteins isolated from Y. pestis strains induced (lanes +) or not induced (lanes −) with 1 mM IPTG. The location of the various Ail/Lom family proteins is indicated by an arrow. The positions of molecular mass standards (in kilodaltons) are indicated on the left. The strains used were Y. pestis strains KIM8-E (parent), KIM8-E Δ4, and KIM8-E Δ4 containing pTRC-y2034, pTRC-y2446, pTRC-Ail, or pTRC-y1682. mw std, molecular mass standards. (B) Survival of the parent strain, the Δ4 strain, and the Δ4 strain carrying plasmid pTRC-y2034, pTRC-y2446, pTRC-Ail, or pTRC-y1682 in 80% NHS. Bacterial cultures were grown at 26°C and induced or not induced with 1 mM IPTG 3 h prior to harvest. Bacteria (∼5 × 10⁶ cells) were incubated with 80% NHS or HIS for 1 h at 37°C. Aliquots were diluted and plated on TBA plates at 30°C. The percent survival was calculated as follows: average number of bacteria that survived exposure to NHS at 1 h/number of bacteria that survived exposure to HIS at 1 h × 100.

FIG. 6.

Ail is required for survival in sera obtained from many diverse mammals but not for survival in mouse serum: survival of the parent (wt) and Δail strains of Y. pestis in 80% animal sera. Bacteria grown overnight at 26°C (∼5 × 10⁶ bacteria) were incubated with 80% animal serum or HIS for 1 h at 37°C. Aliquots were diluted and plated on TBA plates at 30°C. The percent survival was calculated as follows: average number of bacteria that survived exposure to NHS at 1 h/number of bacteria that survived exposure to HIS at 1 h × 100.

FIG. 7.

Ail is required for efficient biofilm-independent killing of C. elegans. Groups of 40 C. elegans N2 young adults were exposed to Y. pestis KIM8-E (parent), KIM8-E Δy2446, KIM8-E Δy2034, KIM8-E Δy1324 (Δail), Δy1324 (Δail)/pAil, KIM8-E Δ4, KIM8-E Δ4/pAil, and Δy1682. Animal survival was plotted using the PRISM computer program.

FIG. 8.

Ail is not required for infection or blockage of fleas. (A) Colonization of fleas after an infected-blood meal. (B) Flea blockage during a 4-week period following a single infected-blood meal. (C) Y. pestis CFU associated with infected fleas on day 0 (week 0) and day 28 (week 4). wt, parent strain KIM6+; Δail, strain KIM6+ Δail.