RfaH promotes the ability of the avian pathogenic Escherichia coli O2 strain E058 to cause avian colibacillosis

Abstract

Avian pathogenic Escherichia coli (APEC) infection causes avian colibacillosis, which refers to any localized or systemic infection, such as acute fatal septicemia or subacute pericarditis and airsacculitis. The RfaH transcriptional regulator in E. coli is known to regulate a number of phenotypic traits. The direct effect of RfaH on the virulence of APEC has not been investigated yet. Our results showed that the inactivation of rfaH significantly decreased the virulence of APEC E058. The attenuation was assessed by in vivo and in vitro assays, including chicken infection assays, an ingestion and intracellular survival assay, and a bactericidal assay with serum complement. The virulence phenotype was restored to resemble that of the wild type by complementation of the rfaH gene in trans. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the deletion of rfaH correlated with decreased virulence of the APEC E058 strain.

Fig 1

Detection of tatD, rfaH, and ubiD gene expression in E058 and E058ΔrfaH by RT-PCR. The RT-PCRs were performed using the following templates: cDNA derived from total RNA of E058 (lanes 1) and E058ΔrfaH (lanes 4) and genomic DNA from E058 (lanes 2) and E058ΔrfaH (lanes 5). Reaction sets contained the following primers: for lanes labeled “a,” TF/TR; for lanes labeled “b,” HF/HR; and for lanes labeled “c,” UF/UR. The negative controls (lanes 3 and 6) used total RNA as the template without activation of RT. A 200-bp marker (TaKaRa) was used as the molecular size standard (M).

Fig 2

Bactericidal activities of SPF chicken serum against wild-type strain E058 and an isogenic mutant. HI represents 100% of heat-inactivated SPF chicken serum. The data represent an average of three trials. The error bars indicate standard deviations. The asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.01).

Fig 3

Ingestion of wild-type strain E058, isogenic mutant E058ΔrfaH, and complementation strain ReE058ΔrfaH by HD-11 cells. The values represent the average data from three independent experiments. The error bars indicate standard deviations. **, statistically significant differences between the wild-type and mutant strains (P < 0.01).

Fig 4

Intracellular growth of bacteria in HD-11 chicken macrophages. The intracellular growth rates of wild-type strain E058, the isogenic mutant E058ΔrfaH, and the complementation strain ReE058ΔrfaH were compared over a 24-h period. Intracellular bacterial growth is shown as the change in the number of intracellular bacteria following an additional 6, 12, and 24 h of incubation compared to the primary ingested bacteria (T₀). Standard errors of the mean (SEM) for three independent experiments are shown. The asterisks indicate statistically significant differences (**, P < 0.01).

Fig 5

Colonization and persistence of the wild-type strain E058 (●), E058ΔrfaH (■), and ReE058ΔrfaH (▲) during systemic infection. The data are presented as log₁₀ CFU · g of tissues⁻¹. The horizontal bars indicate the mean log₁₀ CFU · g⁻¹. Each data point represents a sample from an individual chicken. Statistically significant differences as determined by the Mann-Whitney test are indicated by asterisks (**, P < 0.01). The error bars indicate standard deviations.

Fig 6

In vivo competition assays. E058 (●) and mutant E058ΔrfaH (■) were inoculated simultaneously. The data are presented as log₁₀ CFU · g of tissues⁻¹. The horizontal bars indicate the mean log₁₀ CFU · g⁻¹. Each data point represents a sample from an individual chicken. Statistically significant differences in values between E058 and its mutant are indicated by asterisks (**, P < 0. 01). The error bars indicate standard deviations.

Fig 7

Quantitative RT-PCR analysis of associated virulence gene transcription levels in strains E058, E058ΔrfaH, and ReE058ΔrfaH. RNA was isolated from strains cultured in LB. Transcript levels were measured in cDNA preparations from each strain and normalized to the gapA level, and the results are shown as fold changes relative to the wild-type level. The asterisks indicate statistically significant differences (**, P < 0.01). The error bars indicate standard deviations.