Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is responsible for a wide range of clinical symptoms. How this bacterium adapts itself to ever-changing host milieu is still a mystery. Recently, small non-coding RNAs (sRNAs) have received considerable attention for their functions in fine-tuning gene expression at a post-transcriptional level to promote bacterial adaptation. Here we demonstrate that Hfq, an RNA-binding protein, which facilitates interactions between sRNAs and their mRNA targets, is critical for K. pneumoniae virulence. A K. pneumoniae mutant lacking hfq (Δhfq) failed to disseminate into extra-intestinal organs and was attenuated on induction of a systemic infection in a mouse model. The absence of Hfq was associated with alteration in composition of envelope proteins, increased production of capsular polysaccharides, and decreased resistance to H(2)O(2), heat shock, and UV irradiation. Microarray-based transcriptome analyses revealed that 897 genes involved in numerous cellular processes were deregulated in the Δhfq strain. Interestingly, Hfq appeared to govern expression of many genes indirectly by affecting sigma factor RpoS and RpoE, since 19.5% (175/897) and 17.3% (155/897) of Hfq-dependent genes belong to the RpoE- and RpoS-regulon, respectively. These results indicate that Hfq regulates global gene expression at multiple levels to modulate the physiological fitness and virulence potential of K. pneumoniae.

Figure 1. K. pneumoniae hfq.

(A) Genomic organization of hfq gene in K. pneumoniae. In the Δhfq strain, the coding region of hfq was deleted and replaced by a chloramphenicol cassette amplified from pACYC184. The coding region of hfq, represented as a light grey bar, was cloned to pBAD202, under the control of arabinose-inducible promoter, to give the complementation plasmid pYC343. The grey bar indicates the region of hfq together with its native promoter cloned to pACYC184 yielding the complementation plasmid pYC457. Three promoter regions of hfq are indicated as P1, P2, and P3, respectively. (B) Alignment of hfq sequences of the Gram-negative pathogens with their Hfq functionally identified. The highly conserved SM1 and SM2 motifs are indicated as black and grey lines, respectively.

Figure 2. K. pneumoniae virulence attenuated by the absence of hfq.

(A) Bacterial loads in small intestine, colon, and liver determined at 48 h after oral inoculation with suspension containing equal amount of K. pneumoniae CG43S (5×10⁸ CFU) and Δhfq (5×10⁸ CFU). Filled and open circles represent CG43S and Δhfq retrieved from five BALB/c mice, respectively. (B) Survival of K. pneumoniae-infected mice. Groups of five mice were inoculated by intraperitoneal injection with 2×10³ CFU of CG43S (filled diamonds), or with 1×10⁴ CFU of CG43S (filled squares), Δhfq (open circles), ΔrpoS (open diamonds), or ΔrpoE (open squares), and monitored for 14 days. (C) Groups of six mice were inoculated intraperitoneally with bacterial suspension containing equal amount of K. pneumoniae CG43S (1×10³ CFU) and Δhfq (1×10³ CFU). Bacterial loads of CG43S (filled circles) and Δhfq (open circles) in spleen and liver were determined at 6 h post-inoculation. Horizontal bars indicate geometric means. The limit of detection was approximately 10 CFU. Samples which yielded no colonies were plotted having the value as 10 CFU g⁻¹ tissues. Competitive index is defined as Δhfq _output/CG43S_output ÷ Δhfq _input/CG43S_input. The indicated P values were determined using the Student’s t-test.

Figure 3. Functional classification of Hfq-dependent genes.

Microarray-based transcriptome analyses revealed 897 ORFs that are significantly deregulated in Δhfq as compared to that in CG43S. Based on the genome project of K. pneumoniae NTHU-K2044 (NC012731;[24]), the 897 Hfq-dependent genes were grouped into different functional classes. The values represent the percentage of genes affected by Hfq in Δhfq versus CG43S within the respective class. Black bars: up-regulated genes; grey bars: down-regulated genes.

Figure 4. Hfq modulated the production and expression of K2 CPS.

(A) Enhancement of K2 capsular polysaccharides by the deletion of hfq. Capsular polysaccharides were extracted from overnight-cultured K. pneumoniae CG43S, Δhfq, and the complementation strain Δhfq-C2 by the method described previously . The amount of K2 CPS was reflected by the uronic acid content that was determined by the method described from a glucuronic acid standard curve and expressed as micrograms per 10⁹ CFU. The indicated P values were determined using the Student’s t-test. (B) Genomic organization of K2 cps gene cluster which is depicted from that in K. pneumoniae Chedid (NCBI accession no. D21242.1). (C) Fold changes in transcript abundances of K2 cps genes detected by microarray (black bars) and QPCR (grey bars) in Δhfq relative to that in CG43S are indicated. (D) Fold changes in transcript abundances of CPS-regulating genes, rcsA, rcsB, rcsC, and rmpA detected by microarray (black bars) and QPCR (grey bars) in Δhfq relative to that in CG43S are indicated.

Figure 5. Hfq modulated the expression profiles of envelop proteins.

Two hundred micrograms of extracytoplasmic proteins isolated from overnight-cultured K. pneumoniae CG43S (A) or Δhfq (B) were electrophoresized with two-dimension gels. Images of silver-stained gels analyzed with ImageMasterTM 2D Platinum Version 5.0 are shown. L1-3: landmarks for comparison. Proteins whose expression levels are significantly higher in CG43S or Δhfq are indicated with a red cross. (C) Outer membrane proteins were extracted, fractionated by 12% of SDS-PAGE gel, and silver-stained. The portion of the gel with bands corresponding to the major porins, OmpC, OmpF, and OmpA, is presented. (D) Fold changes in transcript abundances of ompK17, ompF, ompC, ompR, and ybfM detected by microarray (black bars) and QPCR (grey bars) in Δhfq relative to that in CG43S are indicated.

Figure 6. Stress tolerance attenuated in K. pneumoniae lacking hfq, rpoS, or rpoE.

Growth of K. pneumoniae CG43S (filled diamonds), Δhfq (open squares), and Δhfq-C2 (filled circles) in (A) LB and (B) M9 media is determined by CFU calculation at indicative time points. Survival of K. pneumoniae CG43S, Δhfq, Δhfq-C2, ΔrpoS, and ΔrpoE, upon the treatment of 200 mM H₂O₂ for 10 minutes (C), 50°C shock for 10 minutes (D), or UV irradiation (1 J/cm²) (E) are determined by CFU calculation and presented as (CFU after treatment/CFU before treatment) ×100%. * P<0.05, determined with the Student’s t-test.

Figure 7. Hfq affected the expression of sigma factors and small RNAs.

(A) Fold changes in transcript abundances of rpoH, rpoD, rpoS, rpoE, and rpoN detected by microarray (black bars) in Δhfq relative to that in CG43S are indicated. (B) Transcripts of rpoS extracted from CG43S, Δhfq, and Δhfq-C1 were detected by Northern blotting with an rpoS-specific biotin-labeled riboprobe. (C) RpoS proteins isolated from the LB-grown stationary-phase cultures of CG43S, Δhfq, and Δhfq-C2 with or without the induction of 0.02% arabinose were detected by Western blotting with rabbit anti-RpoS antibody. The levels of OmpA were detected with rabbit anti-OmpA antibody as a loading control. (D) RpoE proteins isolated from the LB-grown cultures of CG43S, Δhfq, and Δhfq-C1 at indicated time points were detected by Western blotting with rabbit anti-RpoE antibody. (E) Transcripts of MicF extracted from the LB-grown cultures of CG43S and Δhfq at indicated time points were detected by Northern blotting with a MicF-specific biotin-labeled riboprobe.

Figure 8. Schematic representation of possible mechanisms of Hfq-mediated regulation.

(A) sRNA-dependent negative regulation. Hfq-dependent sRNAs bind their target mRNAs with partial complementarity that occlude the ribosome binding sites, prevents ribosome association and thus represses translation. In many cases, translation inhibition can be coupled to RNA degradation. Hfq recruits RNA degradosome and RNA degradation will be achieved at distal sites from the paring region. (B) Positive regulation of rpoS translation. Hfq-dependent sRNAs, such as DsrA, RprA, and RyhA, relieve an inhibitory secondary structure formed by the rpoS mRNA leader sequence. Derepression of rpoS translation increases the availability of RpoS (σ^S) and thus activates the expression of genes belonging to the rpoS regulon. (C) Regulation through protein-protein interactions. Hfq may contact with proteins to affect cellular processes. Hfq can recruit RNA degradosome, consisting of RNaseE, RhlB helicase, enolase, and PNPase, to stimulate the RNA degradation of sRNA-mRNA duplex. Hfq may alter mRNA stability by promoting PAP I-mediated polyadenylation. In the presence of S1 protein, Hfq may interact with RNA polymerase to modulate transcriptional activities. A minor fraction of Hfq, which was found to associate with the nucleoid, may bind curved DNA, affect negative supercoiling, and then coordinate the transcription and translation activities.