Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli

Abstract

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

Figure 1

FimX phase variation of the hyxR promoter region. A. Comparison of FimX and FimB phase variation of T1P (fimS) and hyxR promoter regions. hyxR phase variation is shown by restriction length dimorphism, corresponding to a unique asymmetric NspI cut site in the promoter after PCR amplification. Lanes 1, 2, and 3 indicate UTI89/pBAD33, UTI89/pBAD:fimB, and UTI89/pBAD:fimX, respectively. B. FimB and FimX inversion of the type 1 and hyxR promoters in wt and recombinase-null backgrounds using phase-specific primers and PCR analysis. Promoter orientation is labeled relative to transcriptional state as “OFF” or “ON” as previously determined for T1P and as shown in Fig. 2B for hyxR. Type 1 pili expression was measured by immunoblot against the major subunit, FimA. Lanes represent UTI89 or ΔfimBEX with the following plasmids: 1=pBAD33, 2=pBAD:fimB, and 3=pBAD:fimX. C. Overview of the genomic organization of the fimX locus PAI-X_UTI89 and location of the P_hyxR inverted repeats. Directionality of the open reading frames, representing forward or reverse strand orientation, is shown. The 16 bp inverted repeats (►) for the hyxR 5’ UTR (P_hyxR-IR_proximal and P_hyxR-IR_distal) are shown with the repeat sequence listed below. The NspI (closest to hyxR) and SspI (closest to fimX) restriction sites used to map the approximate region of inversion are indicated by vertical lines. D. Comparison of inverted repeats and flanking sequence upstream of hyxR and T1P. The region shown is known to be important for FimB and FimE binding to fimS (Dove et al., 1996; Gally et al., 1996;Kulasekara et al., 1999; Holden et al., 2007). The conserved 5’ CA dinucleotides of the fimS repeats are shown in white text on a black background.

Figure 2

fimX expression results in bidirectional inversion of the hyxR promoter region independent of IHF and H-NS. A. fimX expression results in bidirectional inversion of hyxR as assayed by phase PCR. Lanes represent ΔfimBEX phase-locked derivatives with the following plasmids: 1=pBAD33, 2=pBAD:fimB, and 3=pBAD:fimX. Results denote representative experiment. B. hyxR transcript measured by qRT-PCR. Transcript levels are shown as relative fold change as determined using the ΔΔ c(t) method and were normalized to the expression of a control gene transcript (16S rRNA). P-values that reached significance, defined as reaching a p-value < 0.05, are indicated as follows: * p-value < 0.05; *** p-value ≤ 0.001. qRT-PCR results represent at least three independent experiments. C. Investigation of FimX and FimB phase inversion of T1P and hyxR promoter region was undertaken in four additional urinary tract isolates: J96, CFT073, ASB1298, and NU14. Results denote representative experiment. D. FimX and FimB phase inversion of the type 1 promoter and hyxR in either UTI89, ΔH-NS, or ΔhimA (IHF-) deletion strains. Results denote representative experiment

Figure 3

HyxR negatively regulates RNI tolerance in vitro and its expression is responsive to nitrosative stress. A. Growth kinetics of UTI89, ΔhyxR, and a constitutive hyxR expressing strain in MES-LB (100 mM, pH 5.0). All strains grew equally well in pH-matched MES-LB. B. Growth kinetics of UTI89/pTrc99a, ΔhyxR/pTrc99a, and a constitutive hyxR expressing strain in MES-LB (100 mM, pH 5.0) + 3 mM ASN. Results represent at least three independent experiments. C. T1P and hyxR promoter orientation in UTI89 (top two panels) post ASN challenge. Bottom panel shows UTI89/pBAD:fimX after arabinose induction of fimX expression, increasing the starting hyxR phase ON population prior to challenge with ASN. Results denote a representative experiment. D. Transcript analysis of hyxR by qRT-PCR in each of the strains +/− ASN challenge. Transcript levels are shown as relative fold change using the ΔΔ c(t) method and were normalized the expression of a control gene transcript (16S rRNA). P-values that reached significance are indicated as follows: ** p-value < 0.01; *** p-value ≤ 0.001. Error bars represent standard deviation. Results are a composite of at least three independent experiments.

Figure 4

HyxR represses transcript of a key bacterial NO detoxification enzyme, HmpA, correlating with decreased nitrite detoxification in vitro. A. hmpA transcript levels were measured by qRT-PCR in the indicated strains +/− exposure to 3mM ASN for 1 h. Transcript levels are shown as relative fold change using the ΔΔ c(t) method and were normalized the expression of a control gene transcript (16S rRNA). P-values that reached significance are indicated as follows: * p-value < 0.05; ** p-value ≤ 0.01. Error bars represent standard deviation. Results are a composite of at least three independent experiments. B. Growth kinetics of UTI89/pTrc99a, Δhmp/pTrc99a, ΔhmpΔhyxR/pTrc99a, and ΔhmpΔhyxR/pTrc:hyxR in MES-LB (100 mM, pH 5.0) + 3 mM ASN. C. Growth kinetics of UTI89/pBAD33, Δhmp/pBAD33, and Δhmp/pBAD:hmp in MES-LB (100 mM, pH 5.0) + 500 µM ASN. D. Growth kinetics of UTI89/pTrc99a/pBAD33, ΔhyxR/pTrc99a/pBAD33, ΔhyxR/pTrc:hyxR/pBAD33, and ΔhyxR/pTrc:hyxR/pBAD:hmp in MES-LB (100 mM, pH 5.0) + 3 mM ASN.E. Experimental design and growth curve for the RNI detoxification assay. Growth was monitored for the duration of the experiment at indicated time-points. F. At T₀ (addition of ASN) and subsequent indicated times, aliquots of the cultures were taken, centrifuged to remove bacteria, and assayed for nitrite levels using Greiss Reagent. A sample of 500 µM ASN without bacteria (control) was included for reference. Results in Panels B, C, and D represent at least three independent experiments.

Figure 5

HyxR negatively regulates intracellular survival in macrophages through RNI-dependent and -independent mechanisms. A. Schematic diagram of the experimental design and treatment regimen. B. Bacterial titers at 24 h post infection. RAW 264.7 cells were pre-treated with 1 mM L-arginine, an NO precursor, or L-NAME, an iNOS-specific inhibitor 1 h prior to infection and for the duration of the experiment. Unless indicated otherwise, statistical comparisons are between UTI89/pTrc99a and all other strains within each treatment arm. Statistical significance indicated as follows: ns = not significant; * p-value < 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001. C. Relative fitness of different strains under high NO conditions by assessing the ratio of the bacterial burden at 24 h for each strain between a high NO condition (L-arginine) and a low NO condition (L-NAME). D. Cell-free supernatant nitrite levels were measured from the supernatant of infected RAW 264.7 cells using Greiss Reagent (see Experimental Protocols for detailed methods). Unless indicated otherwise, statistical comparisons are between UTI89/pTrc99a and all other strains within each treatment arm. Asterisk (***) represents a p-value ≤ 0.001. E. Nitrite levels were measured from the supernatant of RAW 264.7 cells infected with heat-killed (80°C for 15 min) or arrested (inclusion of 20 µg ml⁻¹ chloramphenicol to inhibit protein synthesis) bacteria using Greiss Reagent. No comparisons reached statistical significance. Abbreviations: ND, not detected.

Figure 6

ExPEC actively suppresses NO production by IFN-activated macrophages, and HyxR negatively regulates intracellular survival in IFNγ-activated macrophages through RNI-dependent and -independent mechanisms. A. Schematic diagram of the experimental design and treatment regimen. B and C. Bacterial titers from 24 h RAW 264.7 cell infections. Cells were not treated (B) or treated with 1 ng ml-1 IFNγ (C) 18 h prior to infection. Unless indicated otherwise, statistical comparisons are between UTI89/pTrc99a and all other strains. Statistical significance indicated as follows: ns = not significant; * p-value < 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001. Error bars indicate 1 standard deviation. D. Relative fitness of different strains in activated macrophages by assessing the ratio of the bacterial burden at 24 h for each strain between IFNγ-activated (C) and unactivated (B) macrophages. E. Nitrite levels were measured in the supernatant of infected RAW 264.7 cells using Greiss Reagent. Unless indicated otherwise, statistical comparisons are between UTI89/pTrc99a and all other strains within each treatment arm. Statistical significance indicated as follows: * p-value < 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001. Error bars indicate standard deviation. Abbreviations: ND, not detected.