RfaH contributes to maximal colonization and full virulence of hypervirulent Klebsiella pneumoniae

Abstract

Hypervirulent K. pneumoniae (hvKp) have emerged as clinically important pathogens, posing a serious threat to human health. RfaH, a transcriptional elongation factor, has been regarded as implicated in facilitating the transcription of long virulence operons in certain bacterial species. In K. pneumoniae, RfaH plays a vital role in promoting CPS synthesis and hypermucoviscosity, as well as mediating bacterial fitness during lung infection. In this study, we aim to conduct a systematic investigation of the roles of rfaH in the survival, dissemination, and colonization of hvKp through in vitro and in vivo assays. We found that bacterial cells and colonies displayed capsule -deficient phenotypes subsequent to the deletion of rfaH in K. pneumoniae NTUH-K2044. We confirmed that rfaH is required for the synthesis of capsule and lipopolysaccharide (LPS) by positively regulating the expression of CPS and LPS gene clusters. We found that the ΔrfaH mutant led to a significantly decreased mortality of K. pneumoniae in a mouse intraperitoneal infection model. We further demonstrated that the absence of rfaH was associated with slower bacterial growth under conditions of low nutrition or iron limitation. ΔrfaH displayed reduced survival rates in the presence of human serum. Besides, the engulfment of the ΔrfaH mutant was significantly higher than that of NTUH-K2044 by macrophages in vivo, indicating an indispensable role of RfaH in the phagocytosis resistance of hvKp in mice. Both mouse intranasal and intraperitoneal infection models revealed a higher bacterial clearance rate of ΔrfaH in lungs, livers, and spleens of mice compared to its wild type, suggesting an important role of RfaH in the bacterial survival, dissemination, and colonization of hvKp in vivo. Histopathological results supported that RfaH contributes to the pathogenicity of hvKp in mice. In conclusion, our study demonstrates crucial roles of RfaH in the survival, colonization and full virulence of hvKp, which provides several implications for the development of RfaH as an antibacterial target.

Keywords: anti-phagocytosis; capsule; hypervirulent Klebsiella pneumoniae; rfaH; virulence.

Figure 1

Construction and morphology characteristics of ΔrfaH. (A) Alignment of amino acid sequences of RfaH proteins from Klebsiella quasipneumoniae (Accession no. WP_131081528), Escherichia coli (MWO99696), Salmonella enterica (EEO3944103), and Yersinia pestis (WP_016594782). Analysis was performed using the ESPript 3.x (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Secondary structures of Kp_RfaH predicted by SWISS-MODEL are indicated by coils for -helices and arrows for -strands. The background of identical residues is marked in red, and homologous residues are highlighted in red. (B) Representative colony phenotypes on the blood agar plates and the optical microscope examination (Gram-staining, 100×10) of NTUH-K2044, ΔrfaH, and ΔrfaH-comp. (C) Transmission electron microscope images of NTUH-K2044 and ΔrfaH. The red arrow indicates the capsule. (D) The string test. The viscous string of NTUH-K2044 and ΔrfaH-comp were positive, measuring >5 mm; the viscous string of ΔrfaH was negative, measuring <5 mm. (E) HMV assay by low-speed centrifugation. Data represent the means of at least three independent experiments, and error bars represent the standard error of the mean. Student’s t-test was performed for statistical analyses, ****P < 0.0001.

Figure 2

ΔrfaH produces less CPS and LPS. (A) Quantification of the capsule by the uronic acid assay. A representative result of this experiment is shown above the bar chart. (B) The transcription levels of genes associated with capsule production (wzi, manC, and rcsA) in NTUH-K2044 and ΔrfaH. (C) Quantification of LPS with phenol-sulfuric acid method; (D) The transcription levels of genes associated with LPS O-antigen (wbbY, glf, and wzt), and core lipopolysaccharide (waaA, and waaF) in NTUH-K2044 and ΔrfaH. Transcript abundance was measured using the 2^-ΔΔCt method with 16s rRNA as a reference gene, and was normalized to the NTUH-K2044. Data represent the mean of at least three independent experiments (in triplicate), and error bars represent the standard error of the mean. P values were calculated using student’s t-tests for statistical analyses, *P < 0.05, **P < 0.01, ****P<0.0001.

Figure 3

ΔrfaH is significantly attenuated in virulence. Survival curves of mice intraperitoneally inoculated with 1 × 10⁵ CFU of NTUH-K2044 or ΔrfaH show that the survival rate of ΔrfaH-infected mice was significantly enhanced (P < 0.0001, by a log-rank [Mantel-Cox] test).

Figure 4

Growth ability and stress resistance assays. (A) Growth curves of NTUH-K2044, ΔrfaH, and ΔrfaH-comp in LB broth. The growth ability of ΔrfaH was comparable to the wild-type strain. (B) Growth curves of NTUH-K2044, ΔrfaH, and ΔrfaH-comp in M9 broth. ΔrfaH exhibited significantly slower growth since 4 h when compared to NTUH-K2044 (P<0.0001), and the maximum bacterial concentration in the stationary phase of ΔrfaH was significantly lower than that of NTUH-K2044 (P<0.0001). (C) Growth curves of NTUH-K2044, ΔrfaH and ΔrfaH-comp in LB broth in the presence of 100 μM, 200 μM, 400 μM or 800 μM 2,2’- dipyridine. ΔrfaH exhibited similar growth ability to NTUH-K2044 in the presence of 100 μM 2,2’-dipyridine, while a significant decreased growth of ΔrfaH was observed in the presence of 200 μM, 400 μM or 800 μM (Overall, P<0.05). (D) The oxidative stress assays. ΔrfaH showed increased resistance to 10 mM H₂O₂. (E) The osmotic stress assays. ΔrfaH showed increased resistance to 0.3 M potassium chloride. Data represent the mean of at least three independent experiments (in triplicate), and error bars represent the standard error of the mean. P values were calculated using student’s t-tests for statistical analyses, **P<0.01, ****P<0.0001.

Figure 5

rfaH deletion improves biofilm formation. (A) Quantification of biofilm formation by crystal violet staining in 96-wells plate. This experiment was performed for three independent times, with six repetitions. A representative result is shown above the bar chart. (B) Continuous monitoring of the biofilm formation for 36 h. (C) The transcription level of genes associated with biofilm formation (mrkA, mrkH, and pgaC). Transcript abundance was measured using the 2^-ΔΔCt method with 16s rRNA as a reference gene, and was normalized to the NTUH-K2044. Data represent the mean of at least three independent experiments (in triplicate), and error bars represent the standard error of the mean. P values were calculated using student’s t-tests for statistical analyses, *P < 0.05, **P<0.01.

Figure 6

rfaH deletion reduces serum and phagocytosis resistance. (A) The serum bactericidal assays. ΔrfaH showed survival defects in 75% serum. Data represent the mean of at least three independent experiments (in triplicate), and error bars represent the standard error of the mean. (B) in vivo phagocytosis assays. Mice (five per group) were intranasally inoculated with 1 × 10⁸ CFU of NTUH-K2044 or ΔrfaH, respectively. A significantly increased engulfment of ΔrfaH by mouse macrophages was observed (P=0.0079). Each symbol represents 1 animal and error bars represent the standard error of the mean. P values were calculated using student’s t-tests for statistical analyses, **P<0.01.

Figure 7

rfaH deletion decreases bacterial dissemination and colonization in mice by the intranasal route. Mice were intranasally inoculated with 1 × 10⁵ CFU of NTUH-K2044 or ΔrfaH, respectively. At the indicated times, mice were euthanized, and the lungs, spleens and livers were homogenized and plated for bacterial enumeration. (A) The organ burden assays after intranasal infection for 6 h, 24 h, and 48 h. Each symbol represents 1 animal, and short bars represent geometric means of each group. The Mann-Whitney test was used for statistical analyses, *P<0.05. ND, not detected. (B) Histopathology of lungs, spleens, and livers in mice after intranasal infection for 24 h.

Figure 8

rfaH deletion decreases bacterial colonization and pathogenicity in mice by the intraperitoneal route. Mice were intraperitoneally inoculated with 1 × 10⁴ CFU of NTUH-K2044 and ΔrfaH, respectively. At the indicated times, mice were euthanized, and the lungs, spleens and livers were homogenized and plated for bacterial enumeration. (A) The organ burden assays after intraperitoneal infection for 6 h, 24 h, and 48 h. Each symbol represents 1 animal, and short bars represent geometric means of each group. The Mann-Whitney test was used for statistical analyses, *P<0.05. ND, not detected. (B) Histopathology of the lungs, spleens, and livers in mice after intraperitoneal infection for 24 h.